Monitoring and tracking the spread of SARS-CoV-2 in Asturias, Spain.

Mutational analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can quantify the relative importance of variants over time, enable dominant mutations to be identified, and facilitate near real-time detection, comparison and tracking of evolving variants. SARS-CoV-2 in Asturias, an autonomous community of Spain with a large ageing population, and high levels of migration and tourism, was monitored and tracked from the beginning of the pandemic in February 2020 until its decline and stabilization in August 2021, and samples were characterized using whole genomic sequencing and single nucleotide polymorphisms. Data held in the GISAID database were analysed to establish patterns in the appearance and persistence of SARS-CoV-2 strains. Only 138 non-synonymous mutations occurring in more than 1 % of the population with SARS-CoV-2 were found, identifying ten major variants worldwide (seven arose before January 2021), 19 regional and one local. In Asturias only 17 different variants were found. After vaccination, no further regional major variants were found. Only half of the defined variants circulated and no new variants were generated, indicating that infection control measures such as rapid diagnosis, isolation and vaccination were efficient.

Comparison of in-house SARS-CoV-2 genome extraction procedures. A need for COVID-19 pandemic.

Among the methods used to diagnose COVID-19, those based on genomic detection by q(RT)-PCR are the most sensitive. To perform these assays, a previous genome extraction of the sample is required. The dramatic increase in the number of SARS-CoV-2 detection assays has increased the demand for extraction reagents hindering the supply of commercial reagents. Homemade reagents and procedures could be an alternative. Nasopharyngeal samples were extracted by seven different methods as well as the automatic method MagNaPure96, to detect SARS-CoV-2. All protocols show sensitivity higher than 87 %, in comparison with reference method, for detecting SARS-CoV-2 as well as human ß- globin. Our results support that these procedures, using common and cheap reagents, are effective to extract RNA (from SARS-CoV-2) or DNA (from human ß-globin) genome from nasopharyngeal swabs. Furthermore, these procedures could be easily adopted by routine diagnostic laboratories to implement detection methods to help to fight against COVID-19 pandemic.

Capillary electrophoresis of PCR fragments with 5´-labelled primers for testing the SARS-Cov-2.

Due to the huge demand for SARS-Cov-2 determination,alternatives to the standard qtPCRtestsare 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.We isolated the naso-pharingealRNA 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.The two SARS-Cov-2 fragments were successfully amplified in the positive samples, while the negative samples did not render fluorescent peaks. In conclusion, we 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 h. By combining a standard PCR with capillary electrophoresis our approach would overcome the limits imposed to many labs by the qtPCR and increase the testing capacity.

Tularemia: diagnosis of an unexpected oculoglandular case in a non-endemic area by universal PCR / Tularemia: diagnóstico de un caso oculoglandular inesperado en un área no endémica mediante PCR universal

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Diagnóstico de meningitis/encefalitis en UCI con sistema de PCR múltiple. ¿Es tiempo de cambio? / Meningitis/Encephalitis diagnosis in ICU using Multiplex PCR system: is it time of change?

OBJETIVO: Evaluar el impacto clínico de la PCR-múltiple FilmArray(R) panel Meningitis/Encefalitis en el diagnóstico de infecciones del sistema nervioso central y comparar los resultados obtenidos y el tiempo necesario hasta el diagnóstico con las técnicas microbiológicas convencionales. PACIENTES Y MÉTODOS: Estudio prospectivo observacional en una Unidad de Cuidados Intensivos (UCI) de adultos de un hospital de tercer nivel. Se realizó punción lumbar a todos los pacientes y en el LCR extraído se realizó FilmArray(R) panel de meningitis /encefalitis, estudio citoquímico, Gram y cultivos microbiológicos convencionales. RESULTADOS: 21 pacientes ingresados con sospecha de Meningitis/Encefalitis. Edad: mediana 58,4 años (RIQ 38,1-67,3), APACHE II: mediana 18 (RIQ 12-24). La mediana de estancia en UCI fue de 4 días (RIQ 2-6) y la hospitalaria de 17 días (RIQ 14-28). Mortalidad 14,3%. Se estableció un diagnóstico clínico final de Meningitis/Encefalitis en 16 pacientes, con diagnóstico etiológico en 12 casos (75%). La etiología más frecuente fue Streptococcus pneumoniae (8 casos). FilmArray(R) permitió diagnóstico etiológico en 3 casos con cultivo negativo y el resultado implicó cambios en el tratamiento antibiótico de 7 de los 16 pacientes (43,8%). Para la totalidad de pacientes, FilmArray(R) presentó una sensibilidad y especificidad del 100% y 90% respectivamente. La mediana de tiempo hasta la obtención del resultado de FilmArray(R) fue de 2,9 horas (RIQ 2,1-3,8) y del cultivo incluyendo antibiograma 45,1 horas (RIQ 38,9-58,7). CONCLUSIONES: FilmArray(R) panel Meningitis/Encefalitis realiza un diagnóstico etiológico más precoz que los cultivos convencionales, muestra una mayor sensibilidad y permite realizar un tratamiento antimicrobiano dirigido

OBJECTIVE: To evaluate the clinical impact of Meningitis/Encephalitis FilmArray(R) panel for the diagnosis of cerebral nervous system infection and to compare the results (including time for diagnosis) with those obtained by conventional microbiological techniques. PATIENTS AND METHODS: A prospective observational study in an Intensive Care Unit of adults from a tertiary hospital was carried out. Cerebrospinal fluid from all patients was taken by lumbar puncture and assessed by the meningitis/encephalitis FilmArray(R) panel ME, cytochemical study, Gram, and conventional microbiological cultures. RESULTS: A total of 21 patients admitted with suspicion of Meningitis/Encephalitis. Median age of patients was 58.4 years (RIQ 38.1-67.3), median APACHE II 18 (RIQ 12-24). Median stay in ICU and median hospital stay was 4 (RIQ 2-6) and 17 days (RIQ 14-28), respectively. The overall mortality was 14.3%. A final clinical diagnosis of meningitis or encephalitis was established in 16 patients, obtaining the etiological diagnosis in 12 of them (75%). The most frequent etiology was Streptococcus pneumoniae (8 cases). FilmArray(R) allowed etiological diagnosis in 3 cases in which the culture had been negative, and the results led to changes in the empirical antimicrobial therapy in 7 of 16 cases (43.8%). FilmArray(R) yielded a global sensitivity and specificity of 100% and 90%, respectively. The median time to obtain results from the latter and conventional culture (including antibiogram) was 2.9 hours (RIQ 2.1-3.8) and 45.1 hours (RIQ 38.9-58.7), respectively. CONCLUSIONS: The Meningitis/Encephalitis FilmArray(R) panel was able to establish the etiologic diagnosis faster than conventional methods. Also, it achieved a better sensitivity and led to prompt targeted antimicrobial therapy

Capnocytophaga canimorsus como causa de sepsis y meningitis en paciente inmunodeprimido / Capnocytophaga canimorsus as a cause of sepsis and meningitis in immunosuppressed patient

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Infección urinaria por Lelliottia amnigena (Enterobacter amnigenus): un patógeno infrecuente / No disponible

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