Postmortem Antigen-Detecting Rapid Diagnostic Tests to Predict Infectivity of SARS-CoV-2-Associated Deaths.

We investigated the infectivity of 128 severe acute respiratory disease coronavirus 2-associated deaths and evaluated predictive values of standard diagnostic procedures. Maintained infectivity (20%) did not correlate with viral RNA loads but correlated well with anti-S antibody levels. Sensitivity >90% for antigen-detecting rapid diagnostic tests supports their usefulness for assessment.

Comparison of real-time and droplet digital PCR to detect and quantify SARS-CoV-2 RNA in plasma.

BACKGROUND: The presence of SARS-CoV-2 RNA in plasma has been linked to disease severity and mortality. We compared RT-qPCR to droplet digital PCR (ddPCR) to detect SARS-CoV-2 RNA in plasma from COVID-19 patients (mild, moderate, and critical disease). METHODS: The presence/concentration of SARS-CoV-2 RNA in plasma was compared in three groups of COVID-19 patients (30 outpatients, 30 ward patients and 30 ICU patients) using both RT-qPCR and ddPCR. Plasma was obtained in the first 24h following admission, and RNA was extracted using eMAG. ddPCR was performed using Bio-Rad SARS-CoV-2 detection kit, and RT-qPCR was performed using GeneFinder™ COVID-19 Plus RealAmp Kit. Statistical analysis was performed using Statistical Package for the Social Science. RESULTS: SARS-CoV-2 RNA was detected, using ddPCR and RT-qPCR, in 91% and 87% of ICU patients, 27% and 23% of ward patients and 3% and 3% of outpatients. The concordance of the results obtained by both methods was excellent (Cohen's kappa index = 0.953). RT-qPCR was able to detect 34/36 (94.4%) patients positive for viral RNA in plasma by ddPCR. Viral RNA load was higher in ICU patients compared with the other groups (P < .001), by both ddPCR and RT-qPCR. AUC analysis revealed Ct values (RT-qPCR) and viral RNA load values (ddPCR) can similarly differentiate between patients admitted to wards and to the ICU (AUC of 0.90 and 0.89, respectively). CONCLUSION: Both methods yielded similar prevalence of RNAemia between groups, with ICU patients showing the highest (>85%). RT-qPCR was as useful as ddPCR to detect and quantify SARS-CoV-2 RNAemia in plasma.

Evaluation of three commercial assays for SARS-CoV-2 molecular detection in upper respiratory tract samples.

The increasing COVID-19 widespread has created the necessity to assess the diagnostic accuracy of newly introduced (RT-PCR based) assays for SARS-CoV-2 RNA detection in respiratory tract samples. We compared the results of the Allplex™ 2019-nCoV assay with those of the Simplexa™ COVID-19 Direct assay and the Quanty COVID-19 assay, respectively, all performed on 125 nasal/oropharyngeal swab samples of patients with COVID-19 suspicion. Fifty-four samples were positive, and 71 were negative with the Allplex™ assay, whereas 47 of 54 samples were also positive with the Simplexa™ assay. The Quanty assay detected 55 positive samples, including the 54 positive samples with the Allplex™ assay and 1 sample that was Allplex™ negative but Simplexa™ positive. Using a consensus result criterion as the reference standard allowed to resolve the eight samples with discordant results (one Allplex™ negative and seven Simplexa™ negative) as truly false negative. Interestingly, a Spearman's negative association was found between the viral RNA loads quantified by the Quanty assay and the CT values of RT PCRs performed with either the Allplex™ assay or the Simplexa™ assay. However, the strength of this association was higher for the Allplex™ assay (N gene, ρ = - 0.92; RdRP gene, ρ = - 0.91) than for the Simplexa™ assay (ORF1ab gene, ρ = - 0.65; S gene, ρ = - 0.80). The Allplex™ 2019-nCoV, the Simplexa™ COVID-19 Direct, and the Quanty COVID-19 assays yielded comparable results. However, the role these assays might play in future clinical practice warrants larger comparison studies.

SARS-CoV-2 Virus Culture and Subgenomic RNA for Respiratory Specimens from Patients with Mild Coronavirus Disease.

We investigated 68 respiratory specimens from 35 coronavirus disease patients in Hong Kong, of whom 32 had mild disease. We found that severe acute respiratory syndrome coronavirus 2 and subgenomic RNA were rarely detectable beyond 8 days after onset of illness. However, virus RNA was detectable for many weeks by reverse transcription PCR.

Viral RNA load in plasma is associated with critical illness and a dysregulated host response in COVID-19.

BACKGROUND: COVID-19 can course with respiratory and extrapulmonary disease. SARS-CoV-2 RNA is detected in respiratory samples but also in blood, stool and urine. Severe COVID-19 is characterized by a dysregulated host response to this virus. We studied whether viral RNAemia or viral RNA load in plasma is associated with severe COVID-19 and also to this dysregulated response. METHODS: A total of 250 patients with COVID-19 were recruited (50 outpatients, 100 hospitalized ward patients and 100 critically ill). Viral RNA detection and quantification in plasma was performed using droplet digital PCR, targeting the N1 and N2 regions of the SARS-CoV-2 nucleoprotein gene. The association between SARS-CoV-2 RNAemia and viral RNA load in plasma with severity was evaluated by multivariate logistic regression. Correlations between viral RNA load and biomarkers evidencing dysregulation of host response were evaluated by calculating the Spearman correlation coefficients. RESULTS: The frequency of viral RNAemia was higher in the critically ill patients (78%) compared to ward patients (27%) and outpatients (2%) (p < 0.001). Critical patients had higher viral RNA loads in plasma than non-critically ill patients, with non-survivors showing the highest values. When outpatients and ward patients were compared, viral RNAemia did not show significant associations in the multivariate analysis. In contrast, when ward patients were compared with ICU patients, both viral RNAemia and viral RNA load in plasma were associated with critical illness (OR [CI 95%], p): RNAemia (3.92 [1.183-12.968], 0.025), viral RNA load (N1) (1.962 [1.244-3.096], 0.004); viral RNA load (N2) (2.229 [1.382-3.595], 0.001). Viral RNA load in plasma correlated with higher levels of chemokines (CXCL10, CCL2), biomarkers indicative of a systemic inflammatory response (IL-6, CRP, ferritin), activation of NK cells (IL-15), endothelial dysfunction (VCAM-1, angiopoietin-2, ICAM-1), coagulation activation (D-Dimer and INR), tissue damage (LDH, GPT), neutrophil response (neutrophils counts, myeloperoxidase, GM-CSF) and immunodepression (PD-L1, IL-10, lymphopenia and monocytopenia). CONCLUSIONS: SARS-CoV-2 RNAemia and viral RNA load in plasma are associated with critical illness in COVID-19. Viral RNA load in plasma correlates with key signatures of dysregulated host responses, suggesting a major role of uncontrolled viral replication in the pathogenesis of this disease.

Expression of TRIM56 gene in SARS-CoV-2 variants and its relationship with progression of COVID-19.

Aim: The present study aimed to determine a correlation between differential TRIM56 expression levels and severe infections of COVID-19 between the Alpha, Delta and Omicron BA.5 variants. Materials & methods: This study was performed on 330 COVID-19 patients, including 142 with severe and 188 with mild infections, as well as 160 healthy controls. The levels of TRIM56 gene expression were determined using a qPCR. Results: TRIM56 gene showed significantly lower mRNA expression in the severe and mild groups compared with healthy individuals. Our finding indicated the high and low reduction of TRIM56 mRNA expression in Delta and Omicron BA.5 variant, respectively. Conclusion: Further research is needed to characterize the impact of TRIM proteins on the severity of COVID-19.

Scientists looked at a protein called TRIM that helps fight viruses to see if a specific TRIM protein, TRIM56, was linked to how poorly people became with COVID-19. The study looked at the blood samples of 330 patients and found that COVID-19 patients had less TRIM56 than healthy people, especially those who were particularly ill.

Viral Load as a Factor Affecting the Fatality of Patients Suffering from Severe Fever with Thrombocytopenia Syndrome.

The clinical characteristics and the effect of viral RNA loads on fatality in 56 patients with severe fever with thrombocytopenia syndrome (SFTS) were analyzed. The non-survival group (12 patients) demonstrated a significantly higher mean age (77 years) than the survival group (44 patients, 65 years) (p = 0.003). The survival rates were 91.7% and 8.3% in patients with Ct values ≥30 and differed significantly (p = 0.001) in the survival and non-survival groups, respectively. The survival rates were 52.4% and 47.6% in patients with viral copy numbers ≥10,000 and 94.3% and 5.7% in patients with viral copy numbers <10,000 in the survival and non-survival groups, respectively (p = 0.001). In a multivariate analysis, viral copy numbers and initial Acute Psychologic Assessment and Chronic Health Evaluation II (APACHE II) scores were identified as the factors affecting fatality (p = 0.015 and 0.011, respectively). SFTS viral RNA loads can be useful markers for the clinical prediction of mortality and survival.

Distribution of hepatitis C virus (HCV) genotypes in a Saudi Arabian hospital during the 2015-2020 period.

Hepatitis C virus (HCV) is the leading cause of chronic liver disease. HCV genotypes and subtypes are important predictors of disease progression and antiviral treatment response. To our knowledge, there had been limited studies of HCV genotypes in Qatif, Saudi Arabia. This study aims to assess the distribution of HCV genotypes in Qatif Central Hospital, Qatif, Saudi Arabia. This is a retrospective study of adult patients with HCV infection between January 2015 and December 2020. Only patients with documented HCV genotyping were included. A total of 356 HCVinfected patients fulfilled the inclusion criteria and were included in further analysis. Of those patients, 179 (50.3%) were males, and most were Saudi (N=347, 97.5%). The median age was 60 years, and 191 (53.7%) were 50-69 years of age. Genotype 2 was present in 118 (33.1%) of the patients, followed by genotype 4 in 92 (25.8%), genotype 1B in 62 (17.4%), and genotype 1A in 37 (10.4%). The study showed that HCV genotype 2 is the predominant variant among chronic HCV patients in the study population. Monitoring the epidemiology of HCV genotypes may provide guidance in treatment decisions.

Severe Acute Respiratory Syndrome Coronavirus 2 Viral RNA Load Status and Antibody Distribution Among Patients and Asymptomatic Carriers in Central China.

This study aimed to monitor severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral loads and specific serum-antibodies (immunoglobulin [Ig] G and M) among confirmed patients and asymptomatic carriers from returning healthy travelers. The throat swabs, sputum, and stool samples from 57 hospitalized coronavirus disease (COVID-19) patients and 8 asymptomatic carriers, among 170 returning healthy travelers were tested using reverse-transcription real-time polymerase chain reaction. SARS-CoV-2 IgM/IgG antibodies were detected via serum chemiluminescence assay. Sequential results showed higher viral RNA loads in the throat, sputum, and stool samples at 3-12 and 6-21 days after symptom onset among severely ill COVID-19 patients. Shorter viral habitation time (1-8 days) was observed in the oropharyngeal site and intestinal tract of asymptomatic carriers. The IgG and IgM response rates were 19/37 (51.4%) and 23/37 (62.6%) among the 29 confirmed patients and 8 asymptomatic carriers, respectively, within 66 days from symptom or detection onset. The median duration between symptom onset and positive IgG and IgM results was 30 (n=23; interquartile range [IQR]=20-66) and 23 (n=19; IQR=12-28) days, respectively. Of 170 returning healthy-travelers to China, 4.7% were asymptomatic carriers (8/170) within 2 weeks, and the IgG and IgM positivity rate was 12.8% (12/94). IgM/IgG-positivity confirmed 3 suspected SARS-CoV-2 cases, despite negative results for SARS-CoV-2 RNA. Compared with other respiratory viral infectious diseases, COVID-19 has fewer asymptomatic carriers, lower antibody response rates, and a longer antibody production duration in recovered patients and the contacted healthy population. This is an indication of the complexity of COVID-19 transmission.

Acute SARS-CoV-2 infection is associated with an increased abundance of bacterial pathogens, including Pseudomonas aeruginosa in the nose.

Research conducted on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis and coronavirus disease 2019 (COVID-19) generally focuses on the systemic host response, especially that generated by severely ill patients, with few studies investigating the impact of acute SARS-CoV-2 at the site of infection. We show that the nasal microbiome of SARS-CoV-2-positive patients (CoV+, n = 68) at the time of diagnosis is unique when compared to CoV- healthcare workers (n = 45) and CoV- outpatients (n = 21). This shift is marked by an increased abundance of bacterial pathogens, including Pseudomonas aeruginosa, which is also positively associated with viral RNA load. Additionally, we observe a robust host transcriptional response in the nasal epithelia of CoV+ patients, indicative of an antiviral innate immune response and neuronal damage. These data suggest that the inflammatory response caused by SARS-CoV-2 infection is associated with an increased abundance of bacterial pathogens in the nasal cavity that could contribute to increased incidence of secondary bacterial infections.

The contrasting role of nasopharyngeal angiotensin converting enzyme 2 (ACE2) transcription in SARS-CoV-2 infection: A cross-sectional study of people tested for COVID-19 in British Columbia, Canada.

BACKGROUND: Angiotensin converting enzyme 2 (ACE2) protein serves as the host receptor for SARS-CoV-2, with a critical role in viral infection. We aim to understand population level variation of nasopharyngeal ACE2 transcription in people tested for COVID-19 and the relationship between ACE2 transcription and SARS-CoV-2 viral load, while adjusting for expression of: (i) the complementary protease, Transmembrane serine protease 2 (TMPRSS2), (ii) soluble ACE2, (iii) age, and (iv) biological sex. The ACE2 gene was targeted to measure expression of transmembrane and soluble transcripts. METHODS: A cross-sectional study of n = 424 "participants" aged 1-104 years referred for COVID-19 testing was performed in British Columbia, Canada. Patients who tested positive for COVID-19 were matched by age and biological sex to patients who tested negative. Viral load and host gene expression were assessed by quantitative reverse-transcriptase polymerase chain reaction. Bivariate analysis and multiple linear regression were performed to understand the role of nasopharyngeal ACE2 expression in SARS-CoV-2 infection. FINDINGS: Analysis showed no association between age and nasopharyngeal ACE2 transcription in those who tested negative for COVID-19 (P = 0•092). Mean relative transcription of transmembrane (P = 0•00012) and soluble (P<0•0001) ACE2 isoforms, as well as TMPRSS2 (P<0•0001) was higher in COVID-19-negative participants than COVID--19 positive ones, yielding a negative correlation between targeted host gene expression and positive COVID-19 diagnosis. In bivariate analysis of COVID-19-positive participants, transcription of transmembrane ACE2 positively correlated with SARS-CoV-2 viral RNA load (B = 0•49, R2=0•14, P<0•0001), transcription of soluble ACE2 negatively correlated (B= -0•85, R2= 0•26, P<0•0001), and no correlation was found with TMPRSS2 transcription (B= -0•042, R2=<0•10, P = 0•69). Multivariable analysis showed that the greatest viral RNA loads were observed in participants with high transmembrane ACE2 transcription (Β= 0•89, 95%CI: [0•59 to 1•18]), while transcription of the soluble isoform appears to protect against high viral RNA load in the upper respiratory tract (Β= -0•099, 95%CI: [-0•18 to -0•022]). INTERPRETATION: Nasopharyngeal ACE2 transcription plays a dual, contrasting role in SARS-CoV-2 infection of the upper respiratory tract. Transcription of the transmembrane ACE2 isoform positively correlates, while transcription of the soluble isoform negatively correlates with viral RNA load after adjusting for age, biological sex, and transcription of TMPRSS2. FUNDING: This project (COV-55) was funded by Genome British Columbia as part of their COVID-19 rapid response initiative.

Initial vaccination and revaccination with Type I PRRS 94881 MLV reduces viral load and infection with porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) causes respiratory distress in pigs, reproductive failure in breeding-age gilts and sows, and can have devastating economic consequences in domestic herds. Several PRRS vaccines are available commercially. This study compared the effectiveness of single-vaccination and revaccination schedules using the PRRS 94881 Type I modified live virus (MLV) vaccine ReproCyc® PRRS EU with no vaccination (challenge control) in protecting against a PRRS virus (PRRSV) challenge in non-pregnant gilts. RESULTS: Data were available from 48 gilts across three groups: a challenge control group (n = 16), which received no vaccination; a revaccination group (n = 16), which received ReproCyc® PRRS EU on Days 0 and 56; and a single vaccination group (n = 16), which received ReproCyc® PRRS EU on Day 56. All gilts were PRRSV RNA-negative (based on reverse transcription and quantitative polymerase chain reaction [RT-qPCR]) and PRRSV seronegative (based on enzyme-linked immunosorbent assay [ELISA]) at Day 0. All gilts were challenged with PRRSV strain 190136 on Day 91.Viral RNA loads in both vaccination groups were significantly reduced compared with the challenge control group on Days 98 (P < 0.0001) and 101 (P < 0.0001), indicating that vaccinated gilts were better able to respond to challenge than unvaccinated gilts. At all timepoints following challenge, mean viral RNA load and the percentage of PRRSV RNA-positive gilts were numerically higher in the single-vaccination group than in the revaccination group; these differences were statistically significant on Day 101 (P = 0.0434). Furthermore, viremia levels after challenge were significantly lower in the revaccination group than in the single-vaccination group based on median area under the curve (AUC) values for viral RNA load from Day 91 to Day 112, suggesting that revaccinated gilts had better protection from viral infection than gilts who received a single vaccination. Protection from viremia did not correlate with the proportion of seropositive gilts on Day 91. In the single-vaccination group, 94% of pigs were seropositive on Day 91 compared with 56% in the revaccination group. Vaccination was well tolerated and no safety concerns were identified. CONCLUSIONS: Both single-vaccination and revaccination with ReproCyc® PRRS EU were effective in reducing PRRSV viremia post-challenge. These findings have important implications for herd management as both the single-vaccination and revaccination schedules protect against PRRSV challenge, with revaccination appearing to provide better protection from viremia than single vaccination.

Viral RNA load and histological changes in tissues following experimental infection with an arterivirus of possums (wobbly possum disease virus).

Tissues from Australian brushtail possums (Trichosurus vulpecula) that had been experimentally infected with wobbly possum disease (WPD) virus (WPDV) were examined to elucidate pathogenesis of WPDV infection. Mononuclear inflammatory cell infiltrates were present in livers, kidneys, salivary glands and brains of WPD-affected possums. Specific staining was detected by immunohistochemistry within macrophages in the livers and kidneys, and undefined cell types in the brains. The highest viral RNA load was found in macrophage-rich tissues. The detection of viral RNA in the salivary gland, serum, kidney, bladder and urine is compatible with transmission via close physical contact during encounters such as fighting or grooming, or by contact with an environment that has been contaminated with saliva or urine. Levels of viral RNA remained high in all tissues tested throughout the study, suggesting that on-going virus replication and evasion of the immune responses may be important in the pathogenesis of disease.

Setting up a SPF chicken model for the pathotyping of West Nile virus (WNV) strains.

Birds play a central role in WNV epidemiology by spreading and amplifying the virus. Increasing numbers of WNV isolates are detected in Europe, and the virulence of these genetically variable isolates is not well characterized for birds. Therefore, we investigated whether SPF chickens could be a valuable avian model for the pathotyping of WNV strains. One-day-old SPF chickens were inoculated subcutaneously (SC) or intracerebrally (IC) with four lineage 1 WNV strains (Is98, It2008, Fr2000 or Kunjin) and were daily clinically monitored for 2 weeks after infection. Additionally, one-day-old SPF chickens were SC inoculated, and one-week-old SPF chickens were SC or IC inoculated with two Euro-Mediterranean isolates, Is98 and Fr2000, to sample blood and feathers at regular time points. These samples were analysed by WN NS2a-specific rRT-PCR and WN NS1 antigen-capture ELISA that were developed for the purpose of this study. Differences in strain virulence were evidenced after IC inoculation of one-day-old SPF chickens, with Is98 eliciting the highest mortality rates and Kunjin the lowest ones, while lethality of Fr2000 and It2008 was intermediate. Neither viral load in sera and feathers nor NS1 antigen in the serum correlated with the differential pathogenicity of Is98 and Fr2000. However, irrespective of the inoculated strain, younger chickens showed higher and longer-lasting viremias than older chickens. In all experimental groups, the detection window for viral RNA in feathers lasted up to 14 dpi. Altogether, the data presented in this study show that WNV strain virulence can be discriminated in a one-day-old SPF chicken model on the basis of mortality rates, while viremia and viral load in feathers appear to be age dependent rather than strain dependent.