Respiratory syncytial virus incidence and typing in the last six seasons in the north of Spain (Asturias). Genetic characterization during the SARS-CoV-2 pandemic.

Respiratory syncytial virus is associated with lower respiratory tract infections. As several types and genotypes can circulate at the same time, genomic characterization is important for timely epidemiological control and treatment measures. In the last 6 seasons (2017-2023), 191 236 nasopharyngeal swabs were processed for respiratory viruses to determine the etiology of acute respiratory infections, describe the incidence and distribution of RSV types and enrich the data of epidemiological molecular studies on RSV in Spain. The incidence of RSV reached 7% in the pre-pandemic season. RSV was most frequent in children under 5 years of age (12.6%), but was also significant in those over 70 years of age (5.63%). The measures taken to control SARS-CoV-2 infection were useful for RSV control and the incidence decreased to 1.8%, but caused a change in the types. Pre-pandemic, the majority circulating types were RSV-B/RSV-B/RSV-A and in the pandemic it was RSV-B/RSV-B. In the last season, RSV-B and RSV-A were detected in the same proportion. Genetic characterization showed three new clades. This has been taken into account to understand the epidemiology as well as the development of therapeutic and preventive strategies.

Detecting, Quantifying, and Isolating Monkeypox Virus in Suspected Cases, Spain.

When a monkeypox virus outbreak began in several parts of the world in May 2022, timely and accurate diagnosis became mandatory. In our laboratory, a real-time quantitative PCR was designed and evaluated in several patient samples and compared with isolation results. Genomic viral load was related to virus viability.

Emergence of New SARS-CoV2 Omicron Variants after the Change of Surveillance and Control Strategy.

In January 2022, there was a global and rapid surge of the Omicron variant of SARS-CoV-2 related to more transmission. This coincided with an increase in the incidence in Asturias, a region where rapid diagnosis and containment measures had limited the circulation of variants. METHODS: From January to June 2022, 34,591 variants were determined by the SNP method. From them, 445 were characterized by the WGS method and classified following pangolin program and phylogenic analysis. RESULTS: The Omicron variant went from being detected in 2438 (78%) samples in the first week of January 2021 to 4074 (98%) in the third week, according to the SNP method. Using the WGS method, 159 BA.1 (35.7%), 256 BA.2 (57.6%), 1 BA.4 (0.2%) and 10 BA.5 (2.2%) Omicron variants were found. Phylogenetic analysis detected that three new sub-clades, BA.2,3.5, BA.2.56 and BF1, were circulating. CONCLUSIONS: The increase in the incidence of SARS-CoV2 caused the circulation of new emerging variants. Viral evolution calls for continuous genomic surveillance.

COVID-19-Associated Pulmonary Aspergillosis in a Tertiary Hospital.

Our study aims to assess the prevalence of CAPA (COVID-19-associated pulmonary aspergillosis) and describe the associated risk factors and their impact on mortality. A prospective study was conducted. We included patients with COVID-19 disease who were admitted to the ICU with a diagnosis of respiratory failur. Mycological culture and other biomarkers (calcofluor staining, LFD, LFA, PCR, GM, and B-D-glucan) were performed. A total of 300 patients were included in the study. Thirty-five patients were diagnosed with CAPA (prevalence 11.7%). During admission, 57 patients died (19%), and, in the group of CAPA patients, mortality was 31.4%. In multivariate analysis, independent risk factors associated with CAPA diagnosis were age (OR: 1.05; 95% CI 1.01-1.09; p = 0.037), chronic lung disease (OR: 3.85; 95% CI 1.02-14.9; p = 0.049) and treatment with tocilizumab during admission (OR: 14.5; 95% 6.1-34.9; p = 0.001). Factors independently associated with mortality were age (OR: 1.06; 95% CI 1.01-1.11; p = 0.014) and CAPA diagnosis during admission (OR: 3.34; 95% CI 1.38-8.08; p = 0.007). CAPA is an infection that appears in many patients with COVID-19 disease. CAPA is associated with high mortality rates, which may be reduced by early diagnosis and initiation of appropriate antifungal therapy, so screening of COVID-19 ARDS (acute respiratory distress syndrome) patients for CAPA is essential.

Angiotensin-converting enzyme (ACE1, ACE2) gene variants are associated with COVID19 severity depending on the hypertension status.

BackgroundThe Angiotensin system is implicated in the pathogenesis of COVID19. First, ACE2 is the cellular receptor for SARS-COv-2, and expression of the ACE2 gene could regulate the individuals susceptibility to infection. In addition, the balance between ACE1 and ACE activity has been implicated in the pathogenesis of respiratory diseases and could play a role in the severity of COVID19. Functional ACE1/ACE2 gene polymorphisms have been associated with the risk of cardiovascular and pulmonary diseases, and could thus also contribute to the outcome of COVID19. MethodsWe studied 204 COVID19 patients (137 non-severe and 67severe-ICU cases) and 536 age-matched controls. The ACE1 insertion/deletion and ACE2rs2285666 polymorphism were determined. Variables frequencies were compared between the groups by logistic regression. We also sequenced the ACE2 coding nucleotides in a group of patients. ResultsSevere COVID19 was associated with hypertension male gender (p<0.001), hypertension (p=0.006), hypercholesterolaemia (p=0.046), and the ACE1-DD genotype (p=0.049). In the multiple logistic regression hypertension (p=0.02, OR=2.26, 95%CI=1.12-4.63) and male gender (p=0.002; OR=3.15, 95%CI=1.56-6.66) remained as independent significant predictors of severity. The ACE2 polymorphism was not associated with the disease outcome. The ACE2 sequencing showed no coding sequence variants that could explain an increased risk of developing COVID19. ConclusionsAdverse outcome of COVID19 was associated with male gender, hypertension, hypercholesterolemia and the ACE1 genotype. The ACE1-I/D was a significant risk factor for severe COVID19, but the effect was dependent on the hypertensive status.

Capillary Electrophoresis of PCR fragments with labelled primers for testing the SARS-Cov-2

BackgroundDue to the huge demand for SARS-Cov-2 determination, alternatives to the standard qtPCR tests are potentially useful for increasing the number of samples screened. Our aim was to develop a direct fluorescent PCR capillary-electrophoresis detection of the viral genome. We validated this approach on several SARS-Cov-2 positive and negative samples. Study designWe isolated the naso-pharingeal RNA from 20 positive and 10 negative samples. The cDNA was synthesised and two fragments of the SARS-Cov-2 were amplified. One of the primers for each pair was 5-end fluorochrome labelled. The amplifications were subjected to capillary electrophoresis in ABI3130 sequencers to visualize the fluorescent peaks. ResultsThe two SARS-Cov-2 fragments were successfully amplified in the positive samples, while the negative samples did not render fluorescent peaks. ConclusionWe describe and alternative method to identify the SARS-Cov-2 genome that could be scaled to the analysis of approximately 100 samples in less than 5 hours. By combining a standard PCR with capillary electrophoresis our approach would overcome the limits imposed to many labs by the qtPCR (lack of reactive and real-time PCR equipment) and increase the testing capacity.