Comparing RNA extraction methods to face the variations in RNA quality using two human biological matrices.

BACKGROUND: Nucleic acids, RNA among them, are widely used in biomedicine and Biotechnology. Because of their susceptibility to degradation by RNases, the handling and extraction process of RNA from cells and tissues require specialized personnel and standardized methods to guarantee high purity and integrity. Due to the diversity of techniques found in the market, a comparative study between different RNA extraction methods is useful to facilitate the best choice for the researcher or in research service platforms such as biobanks to see the traceability of the samples. METHODS AND RESULTS: In this study, we have compared seven different RNA extraction methods: manual (TRIzol™), semiautomated (QIAGEN™, Bio-Rad, Monarch®, and Canvax™), and fully automated (QIAcube™ and Maxwell®) processes, from two biological matrices: human Jurkat T cells and peripheral blood mononuclear cells (PBMC). Results showed marked differences in the RNA quality and functionality according to the method employed for RNA extraction and the matrix used. DISCUSSION: QIAcube™ and semi-automated extraction methods were perceived as the best options because of their lower variability, good functionality, and lower cost (P < 0.001). These data contribute to facilitating researchers or research service platforms (Biobanks) in decision-making practices and emphasize the relevance of the selection of the RNA extraction method in each experimental procedure or traceability study to guarantee both quality standards and its reproducibility.

Cost-effectiveness analysis of dyevert™ Power XT in patients with chronic kidney disease undergoing percutaneous coronary intervention procedures in Spain.

OBJECTIVE: To assess the efficiency of Dyevert™ Power XT compared to the standard clinical practice when used for percutaneous coronary interventions (PCI). METHODS: A Markov model was developed to estimate, over 3-month cycles and a lifetime time horizon, the cumulative costs and health outcomes (life years gained [LYG] and quality-adjusted life years [QALY]) in a hypothetical cohort of 1,000 patients with chronic kidney disease (CKD) 3b-4 and an average age of 72 years. The incidence of contrast-induced acute kidney injury for these patients is 18.89% in routine practice and 7.78% with Dyevert. QALYs were estimated by applying utilities by health state. Transitions between states and utilities were obtained from the literature. Overall all-cause and state-specific mortality were considered. The total cost (€2,022) estimated with the National Health System perspective included cost of the procedure and of CKD management. The parameters were validated by a panel of experts. A discount rate (3% per year) was applied to costs and outcomes. RESULTS: The use of Dyevert yielded more health benefits (34.60 LYG and 5.69 QALYs) compared to the current standard practice (33.11 LYG and 5.38 QALYs). Lifetime cost accumulated at the end of the simulation resulted €30,211/patient with Dyevert and €33,895/patient with current standard clinical practice. CONCLUSIONS: The use of Dyevert™ Power XT resulted dominant option, due to its higher effectiveness and lower cost as compared to standard clinical practice and, therefore, a preferred option in patients with CKD stages 3b-4 undergoing PCI in Spain.

[Tuberculous otitis media: A case presentation and review of european literature]. / Otitis media tuberculosa: Presentación de un caso y revisión de la literatura europea.

Tuberculous otitis media (TOM) is a rare affectation in our environment that represents a challenge in its diagnosis due to the non-specific symptoms that it usually presents. This paper presents our experience in the diagnosis of a case of TOM in a 66-year-old woman with hearing loss and chronic otorrhea of more than 6 months of evolution that did not respond to conventional treatments. In addition, a review of the cases published in the last 20 years (2000-2022) in countries of the European Union (EU) is carried out. The most common symptoms were otorrhea (n=43; 100%), hearing loss (n=37; 86.05%), eardrum perforation (n=19; 44.18%), facial paralysis (n=12, 27,91%) and ear pain (n=13; 30,23%). The most used sample for diagnosis was the biopsy obtained by mastoidectomy (n=34; 79.06%). All patients were given antituberculous therapy for a mean duration of 8.11 months (range, 6-12 months). The most frequent aftereffect was hearing loss (n=28; 65.12%). TOM should be included in the differential diagnosis of chronic suppurative otitis, since early diagnosis and treatment reduce the probability of suffering irreversible sequelae.

[Comparison of the first and sixth waves of the SARS-COV-2 coronavirus pandemic in patients with immune-mediated disease.] / Comparación entre la primera y la sexta ola de la pandemia por el coronavirus SARS-COV-2 en pacientes con enfermedad inmunomediada.

Recent evidence shows that COVID-19 infection does not have a worse prognosis in patients with immune-mediated inflammatory diseases (IMID), although they develop a worse response to vaccination.Objective To compare the incidence of COVID-19 and clinical features in patients with IMID between the first and sixth waves.Method Prospective observational study of two cohorts of IMID patients diagnosed with COVID-19. First cohort March to May 2020, and second cohort December/2021 to February/2022.Sociodemographic and clinical variables were collected and, in the second cohort, COVID-19 vaccination status. Statistical analysis established differences in characteristics and clinical course between the two cohorts.Results In total, 1627 patients were followed up, of whom 77 (4.60%) contracted COVID-19 during the first wave and 184 in the sixth wave (11.3%). In the sixth wave, there were fewer hospitalisations, intensive care unit admissions, and deaths than in the first wave (p=.000) and 180 patients (97.8%) had at least one dose of vaccine.Conclusion Early detection and vaccination have prevented the occurrence of serious complications.

Fatty liver index as a predictor for type 2 diabetes in subjects with normoglycemia in a nationwide cohort study.

Our aim was to evaluate whether fatty liver index (FLI) is associated with the risk of type 2 diabetes (T2DM) development within the Spanish adult population and according to their prediabetes status; additionally, to examine its incremental predictive value regarding traditional risk factors. A total of 2260 subjects (Prediabetes: 641 subjects, normoglycemia: 1619 subjects) from the Di@bet.es cohort study were studied. Socio-demographic, anthropometric, clinical data and survey on habits were recorded. An oral glucose tolerance test was performed and fasting determinations of glucose, lipids and insulin were made. FLI was calculated and classified into three categories: Low (< 30), intermediate (30-60) and high (> 60). In total, 143 people developed diabetes at follow-up. The presence of a high FLI category was in all cases a significant independent risk factor for the development of diabetes. The inclusion of FLI categories in prediction models based on different conventional T2DM risk factors significantly increase the prediction power of the models when all the population was considered. According to our results, FLI might be considered an early indicator of T2DM development even under normoglycemic condition. The data also suggest that FLI could provide additional information for the prediction of T2DM in models based on conventional risk factors.

Incidence of diabetes mellitus in Spain as results of the nation-wide cohort di@bet.es study.

Our aim was to determine the incidence of type 2 diabetes mellitus in a nation-wide population based cohort from Spain (di@bet.es study). The target was the Spanish population. In total 5072 people older than 18 years,were randomly selected from all over Spain). Socio-demographic and clinical data, survey on habits (physical activity and food consumption) and weight, height, waist, hip and blood pressure were recorder. A fasting blood draw and an oral glucose tolerance test were performed. Determinations of serum glucose were made. In the follow-up the same variables were collected and HbA1c was determined. A total of 2408 subjects participated in the follow-up. In total, 154 people developed diabetes (6.4% cumulative incidence in 7.5 years of follow-up). The incidence of diabetes adjusted for the structure of age and sex of the Spanish population was 11.6 cases/1000 person-years (IC95% = 11.1-12.1). The incidence of known diabetes was 3.7 cases/1000 person-years (IC95% = 2.8-4.6). The main risk factors for developing diabetes were the presence of prediabetes in cross-sectional study, age, male sex, obesity, central obesity, increase in weight, and family history of diabetes. This work provides data about population-based incidence rates of diabetes and associated risk factors in a nation-wide cohort of Spanish population.

Polymorphisms within the ARNT2 and CX3CR1 Genes Are Associated with the Risk of Developing Invasive Aspergillosis.

Invasive aspergillosis (IA) is a life-threatening infection that affects an increasing number of patients undergoing chemotherapy or allo-transplantation, and recent studies have shown that genetic factors contribute to disease susceptibility. In this two-stage, population-based, case-control study, we evaluated whether 7 potentially functional single nucleotide polymorphisms (SNPs) within the ARNT2 and CX3CR1 genes influence the risk of IA in high-risk hematological patients. We genotyped selected SNPs in a cohort of 500 hematological patients (103 of those had been diagnosed with proven or probable IA), and we evaluated their association with the risk of developing IA. The association of the most interesting markers of IA risk was then validated in a replication population, including 474 subjects (94 IA and 380 non-IA patients). Functional experiments were also performed to confirm the biological relevance of the most interesting markers. The meta-analysis of both populations showed that carriers of the ARNT2rs1374213G, CX3CR1rs7631529A, and CX3CR1rs9823718G alleles (where the RefSeq identifier appears as a subscript) had a significantly increased risk of developing IA according to a log-additive model (P value from the meta-analysis [PMeta] = 9.8 · 10-5, PMeta = 1.5 · 10-4, and PMeta =7.9 · 10-5, respectively). Haplotype analysis also confirmed the association of the CX3CR1 haplotype with AG CGG with an increased risk of IA (P = 4.0 · 10-4). Mechanistically, we observed that monocyte-derived macrophages (MDM) from subjects carrying the ARNTR2rs1374213G allele or the GG genotype showed a significantly impaired fungicidal activity but that MDM from carriers of the ARNT2rs1374213G and CX3CR1rs9823718G or CX3CR1rs7631529A alleles had deregulated immune responses to Aspergillus conidia. These results, together with those from expression quantitative trait locus (eQTL) data browsers showing a strong correlation of the CX3CR1rs9823718G allele with lower levels of CX3CR1 mRNA in whole peripheral blood (P = 2.46 · 10-7) and primary monocytes (P = 4.31 · 10-7), highlight the role of the ARNT2 and CX3CR1 loci in modulating and predicting IA risk and provide new insights into the host immune mechanisms involved in IA development.

Clinical and microbiologic characteristics of pleuro-pulmonary infection due to Streptococcus intermedius.

The clinical and microbiological characteristics of pleuro-pulmonary infection (PPI) caused by Streptococcus intermedius is described, including 6 cases in the literature and 9 cases handled at the present centre. Out of the 15 patients, 12 were male; mean age at diagnosis was 62.06 ± 15 years. Twelve had risk factors for S. intermedius infection such as alcoholism in 5 (35.7%) patients, periodontal disease in 3 (24.6%) cases, chronic obstructive pulmonary disease in 3 (24.6%), and diabetes mellitus in 2 (14.2%). Cough was present in 12 (80%) patients and chest pain and dyspnea in 9 (60%). The mean diagnosis interval was 34 days. The diagnosis was obtained from pleural fluid aspirate in 13 (86.6%) cases and from biopsy/tissue samples in 2. The most frequently antimicrobials used for treatment were ceftriaxone + levofloxacin. Ten patients cured with a combination of medical and surgical treatment and 2 patients died as a consequence of infection. The incidence of PPI caused by S. intermedius is increasing in our health area; drainage along with antibiotic therapy is recommended for treatment.

Effect of Single Layer Centrifugation Porcicoll (70%, 80% and 90%) or supplementation with reduced glutathione, seminal plasma and bovine serum albumin on frozen-thawed boar sperm.

Selecting the optimal sperm population is essential for success with reproductive techniques. Porcicoll (formerly Androcoll-P) is a colloid formulation for selection of high-quality boar spermatozoa by single layer centrifugation (SLC). To date, most studies have been carried out with fresh semen and large volumes. We carried out 2 experiments to test the use of Porcicoll for thawed boar semen in small volumes. In Experiment 1, cryopreserved semen doses were thawed, split in 200-µL aliquots and layered on 1mL of Porcicoll 70%, 80% or 90%, or buffer without colloid. We assessed sperm recovery (the proportion of the loading dose that appeared in the pellet, %), and the physiology of the selected spermatozoa (flow cytometry: Viability, apoptotic changes, capacitation, mitochondrial activity, intracellular reactive oxygen species). The most suitable proportion was Porcicoll 80%, allowing acceptable sperm recovery (16.9±4.2%, compared to 70% (35.4%±3.0, p<0.001) and 90% (8.2%±3.0, P=0.001), and improved quality (mitochondrial activity: Porcicoll 80%: 77.7±1% vs Control: 60.3±0.7%, P<0.05). In Experiment 2, we compared 3 supplements to Porcicoll 80%: 500mM reduced glutathione (GSH), 20% seminal plasma (SP) and 0.5% bovine serum albumin (BSA). Supplementation with GSH or BSA did not cause relevant changes relative to Control. In contrast, SP induced membrane and acrosomal changes resembling capacitation, which might preclude its use in some applications, and decreased recovery (5.5%±1.9 vs. 24.3%±1.2 Control; P<0.001). However, it could be useful prior to other applications such as in vitro fertilisation. Overall, Porcicoll is an effective colloid for isolating a high-quality population from thawed boar sperm, 80% being a balanced option for good recovery and high quality. Supplements could be useful depending on the proposed use of the spermatozoa.

Clinical and microbiological findings of septic arthritis caused by Haemophilus parainfluenzae.

OBJECTIVES: To report a case of septic arthritis due to H. parainfluenzae and to review the clinical and microbiological characteristics of published case patients. PATIENTS AND METHODS: Data was collected on age, sex, infection localization, underlying risk factors, symptom onset-diagnosis interval, analytical findings, microbiological diagnosis, treatment, outcome, and follow-up of the present patient (presenting with septic arthritis of the pubic symphysis due to H. parainfluenzae) and those identified in a literature analysis. RESULTS: Data of 18 patients, including 17 reported case patients, was collected. Mean age at presentation was 51±9 years. Underlying diseases for septic arthritis were recorded in 11 patients. The infection site was the knee in eight patients, hip and/or acromioclavicular joint in five. Pain was observed in 15 patients and fever in 10; the mean symptom onset-diagnosis interval was 9.4 days. Diagnosis was obtained from synovial fluid aspirate in 12 patients and from blood cultures in four. Susceptibility of H. parainfluenzae strains was reported in 12 cases. Eight patients were treated with cephalosporins and 10 with penicillins. A favorable outcome was observed in 13 patients. CONCLUSIONS: Septic arthritis caused by H. parainfluenzae is a rare entity that requires a high level of suspicion before application of laboratory methods for rapid diagnosis and treatment.

Streptococcus agalactiae: prevention and vaccine development.

Streptococcus agalactiae, group B Streptococcus (SGB), is the most important cause of morbi-mortality among newborn population, and an important pathogen among immunossupressed adult patients. Despite the advances in the treatment and prevention of neonatal infections as a consequence of implementation of national and international recommendations for prevention of infection, there are still some improvements for the final control of the disease. In this sense, the vaccination against SGB could be an effective measure for the prevention of disease in those cases where intrapartum prophylaxis is not useful and in adult patients with risk factors for invasive infection due to SGB. This review summarizes the efforts made until now in order to establish the control of the infection, and brings some information on the current state-of-the art of vaccines against SGB, in which different strategies in their design have been used.

Endoftalmitis endógenas bacterianas. A propósito de 2 casos / Endogenous bacterial endophthalmitis. A report of 2 cases

Casos clínicos: Presentamos 2 casos clínicos de endoftalmitis endógena bacteriana. El primero causado por Streptococcus bovis tras la realización de una colonoscopia, con mala evolución que acabó en evisceración. El segundo debido a un Staphylococcus aureus meticilin resistente procedente de un absceso paraespinal que complicaba una artrodesis, con excelente recuperación visual. Discusión: La endoftalmitis endógena bacteriana es una enfermedad poco frecuente y grave en la que bacterias procedentes de un foco primario llegan por vía sanguínea hasta el ojo. El diagnóstico de sospecha ha de ser lo más precoz posible, basado en la clínica y los antecedentes del paciente. Un tratamiento adecuado y específico con antibióticos intravenosos e intravítreos puede evitar el mal pronóstico visual en algunos casos (AU)

Clinical cases: The cases are presented on 2 patients with bacterial endogenous endophthalmitis. The first one was caused by Streptococcus bovis, developed after colonoscopy, which had a poor outcome and resulted in evisceration. The second case was caused by a methicillin resistant Staphylococcus aureus from an arthrodesis complicated with a para-spinal abscess. It had an excellent visual outcome. Discussion: Bacterial endogenous endophthalmitis is a rare, but serious ocular disease that occurs when bacteria reach the eye via the bloodstream. It requires a very early diagnosis based on the clinical symptoms and patient history. A suitable and specific treatment with intravenous and intravitreal antibiotics may prevent a bad visual prognosis in some cases (AU)

Pleural effusion due to Parvimonas micra. A case report and a literature review of 30 cases.

The clinical and microbiological characteristics of infections caused by Parvimonas micra is described, including 30 cases in the literature and a new case handled at the present centre. Out of the 31 patients, 18 were male; mean age at diagnosis was 65.1 ± 13.0 years. Infection site was the vertebral spine in 14 patients and joints and heart valves in 5 each one; pain was present in all patients with articular localization and in almost all patients with vertebral involvement. The diagnosis was obtained from fluid aspirate or drainage in 13 cases and blood cultures in 11. In 8 cases, molecular techniques were also applied. The most frequently used antimicrobials were clindamycin, penicillin, amoxicillin and ceftriaxone. The outcome was positive with the medical treatment in 28 patients. P. micra infections are uncommon and requires a high index of suspicion.

Endogenous bacterial endophthalmitis. A report of 2cases. / Endoftalmitis endógenas bacterianas. A propósito de 2casos.

CLINICAL CASES: The cases are presented on 2patients with bacterial endogenous endophthalmitis. The first one was caused by Streptococcus bovis, developed after colonoscopy, which had a poor outcome and resulted in evisceration. The second case was caused by a methicillin resistant Staphylococcus aureus from an arthrodesis complicated with a para-spinal abscess. It had an excellent visual outcome. DISCUSSION: Bacterial endogenous endophthalmitis is a rare, but serious ocular disease that occurs when bacteria reach the eye via the bloodstream. It requires a very early diagnosis based on the clinical symptoms and patient history. A suitable and specific treatment with intravenous and intravitreal antibiotics may prevent a bad visual prognosis in some cases.

Helper-dependent adenovirus achieve more efficient and persistent liver transgene expression in non-human primates under immunosuppression.

Helper-dependent adenoviral (HDA) vectors constitute excellent gene therapy tools for metabolic liver diseases. We have previously shown that an HDA vector encoding human porphobilinogen deaminase (PBGD) corrects acute intermittent porphyria mice. Now, six non-human primates were injected in the left hepatic lobe with the PBGD-encoding HDA vector to study levels and persistence of transgene expression. Intrahepatic administration of 5 × 10(12) viral particles kg(-1) (10(10) infective units kg(-1)) of HDA only resulted in transient (≈14 weeks) transgene expression in one out of three individuals. In contrast, a more prolonged 90-day immunosuppressive regimen (tacrolimus, mycophenolate, rituximab and steroids) extended meaningful transgene expression for over 76 weeks in two out of two cases. Transgene expression under immunosuppression (IS) reached maximum levels 6 weeks after HDA administration and gradually declined reaching a stable plateau within the therapeutic range for acute porphyria. The non-injected liver lobes also expressed the transgene because of vector circulation. IS controlled anticapsid T-cell responses and decreased the induction of neutralizing antibodies. Re-administration of HDA-hPBGD at week +78 achieved therapeutically meaningful transgene expression only in those animals receiving IS again at the time of this second vector exposure. Overall, immunity against adenoviral capsids poses serious hurdles for long-term HDA-mediated liver transduction, which can be partially circumvented by pharmacological IS.