Reliable and specific detection of Acanthamoeba spp. in dishcloths using quantitative real-time PCR assay.

Acanthamoeba spp., are ubiquitous protist which belongs to Free-Living Amoeba (FLA) group, is considered as causal agent of side-threatening keratitis or fatal encephalitis among other human infections. Besides, this parasite has been reported as host for other microorganisms important to human health such as Campylobacter spp. or Vibrio spp. among others. This role of Acanthamoeba as pathogen and environmental phagocyte has increased the reports confirming its presence in human related environments, acting as a water quality indicator. Considering the tide relationship between water and kitchen environments, and the high prevalence of Acanthamoeba in water sources, the present study aims to establish a quick and accurate protocol based on DNA extraction and a real time qPCR assay to detect Acanthamoeba spp. in dishcloths. The procedure has been validated by processing 17 used dishcloths. Our findings demonstrated the high sensitivity of the qPCR assay used which was capable of detecting up to one Acanthamoeba from an in vitro contaminated dishcloth. The protocol accurately detected 64.7% of positive samples for Acanthamoeba spp, (in 4 samples DNA concentrations corresponded to 1-102 amoebae). Our findings demonstrate the importance of FLA surveillance by efficient and sensitive methods since one amoeba is capable of colonizing human related food environments such as kitchens sinks and could be a potential source of infection.

Development of an equipment for real-time continuous monitoring of alpha and beta radioactivity in river water.

Different regulations require the monitoring of radioactivity in the environment (e.g., 2013/51/Euratom, Real Decreto 314/2016) to protect the environment and the population from abnormal radioactivity presence caused by natural reasons or discharges or accidents in nuclear installations. Nowadays, the monitoring of α- and ß-emitting radionuclides is performed discontinuously in laboratories due to the difficulties in applying classical techniques to continuous measurements. This limits the number of samples that can be measured per day, produces high costs per analysis, and introduces a significant delay between the moment of contamination and when it is detected. Plastic scintillation microspheres (PSm) represent a new possibility for continuous measurements because water samples can flow through a bed of PSm connected to a pair of photomultipliers (PMTs), allowing continuous monitoring of the activity. This idea is the basis of the Waterrad detector, which can monitor radioactivity at environmental levels in river water. This paper describes the optimization of a detection cell containing PSm, a detection chamber as well as active and passive shielding. In its final set-up, the Waterrad detector presents a background signal of 0.23 (1) cps and detection efficiencies of 1.86(7)·10-5 cps·L·Bq-1 for 3H, 7.4(8)·10-3 cps·L·Bq-1 for 90Sr/90Y and 5.5(5)·10-3 cps·L·Bq-1 for 241Am. The detection limits in the optimum window for a counting time of 5 h were 490 Bq/L for 3H, 2.3 Bq/L for 90Sr/90Y and 3.0 Bq/L for 241Am. These values indicate that Waterrad can be used as an alarm detector for monitoring radioactivity in water at activity levels similar to those of environmental samples, making it suitable for water or waste surveillance involving a high frequency of measurements.

In vitro amoebicidal effects of arabinogalactan-based ophthalmic solution.

The main corneal infections reported worldwide are caused by bacteria and viruses but, recently, the number of Acanthamoeba keratitis (AK) cases has increased. Acanthamoeba genus is an opportunistic free living protozoa widely distributed in environmental and clinical sources, with two life-cycle stages: the trophozoite and the cyst. AK presents as primary symptoms eye redness, epithelial defects, photophobia and intense pain. An early diagnosis and an effective treatment are crucial to avoid blindness or eye removal but, so far, there is no established treatment to this corneal infection. Diverse research studies have reported the efficacy of commercialized eye drops and ophthalmic solutions against the two life cycle stages of Acanthamoeba strains, that usually present preservatives such as Propylene Glycol of Benzalkonium chloride (BAK). These compounds present toxic effects in corneal cells, favouring the inflammatory response in the so sensitive eye tissue. In the present work we have evaluated the efficacy of nine proprietary ophthalmic solutions with and without preservatives (ASDA Dry Eyes Eyedrops, Miren®, ODM5®, Ectodol®, Systane® Complete, Ocudox®, Matrix Ocular®, Alins® and Coqun®) against the two life cycle stages of three Acanthamoeba strains. Our work has demonstrated the high anti-Acanthamoeba activity of Matrix Ocular®, which induces the programmed cell death mechanisms in Acanthamoeba spp. trophozoites. The high efficacy and the absence of ocular toxic effects of Matrix Ocular®, evidences the use of the Arabinogalactan derivatives as a new source of anti-AK compounds.

JavaCyte, a novel open-source tool for automated quantification of key hallmarks of cardiac structural remodeling.

Many cardiac pathologies involve changes in tissue structure. Conventional analysis of structural features is extremely time-consuming and subject to observer bias. The possibility to determine spatial interrelations between these features is often not fully exploited. We developed a staining protocol and an ImageJ-based tool (JavaCyte) for automated histological analysis of cardiac structure, including quantification of cardiomyocyte size, overall and endomysial fibrosis, spatial patterns of endomysial fibrosis, fibroblast density, capillary density and capillary size. This automated analysis was compared to manual quantification in several well-characterized goat models of atrial fibrillation (AF). In addition, we tested inter-observer variability in atrial biopsies from the CATCH-ME consortium atrial tissue bank, with patients stratified by their cardiovascular risk profile for structural remodeling. We were able to reproduce previous manually derived histological findings in goat models for AF and AV block (AVB) using JavaCyte. Furthermore, strong correlation was found between manual and automated observations for myocyte count (r = 0.94, p < 0.001), myocyte diameter (r = 0.97, p < 0.001), endomysial fibrosis (r = 0.98, p < 0.001) and capillary count (r = 0.95, p < 0.001) in human biopsies. No significant variation between observers was observed (ICC = 0.89, p < 0.001). We developed and validated an open-source tool for high-throughput, automated histological analysis of cardiac tissue properties. JavaCyte was as accurate as manual measurements, with less inter-observer variability and faster throughput.

Efficacy of extended infusion of ß-lactam antibiotics for the treatment of febrile neutropenia in haematologic patients: protocol for a randomised, multicentre, open-label, superiority clinical trial (BEATLE).

BACKGROUND: Febrile neutropaenia (FN) is a very common complication in patients with haematological malignancies and is associated with considerable morbidity and mortality. Broad-spectrum antipseudomonal ß-lactam antibiotics (BLA) are routinely used for the treatment of cancer patients with FN. However, the clinical efficacy of BLA may be diminished in these patients because they present with pathophysiological variations that compromise the pharmacokinetic (PK) parameters of these antibiotics. Optimised administration of BLA in prolonged infusions has demonstrated better clinical outcomes in critically ill patients. However, there is a paucity of data on the usefulness of this strategy in patients with FN. The aim of this study is to test the hypothesis that the administration of BLA would be clinically more effective by extended infusion (EI) than by intermittent infusion (II) in haematological patients with FN. METHODS: A randomised, multicentre, open-label, superiority clinical trial will be performed. Patients with haematological malignancies undergoing chemotherapy or haematopoietic stem-cell transplant and who have FN and receive empirical antibiotic therapy with cefepime, piperacillin-tazobactam or meropenem will be randomised (1:1) to receive the antibiotic by EI (during half the time of the dosing interval) in the study group, or by II (30 min) in the control group. The primary endpoint will be clinical efficacy, defined as defervescence without modifying the antibiotic treatment administered within the first 5 days of therapy. The primary endpoint will be analysed in the intention-to-treat population. The secondary endpoints will be pharmacokinetic/pharmacodynamic (PK/PD) target achievement, bacteraemia clearance, decrease in C-reactive protein, overall (30-day) case-fatality rate, adverse events and development of a population PK model of the BLA studied. DISCUSSION: Data on the usefulness of BLA administration in patients with FN are scant. Only three clinical studies addressing this issue have been published thus far, with contradictory results. Moreover, these studies had some methodological flaws that limit the interpretation of their findings. If this randomised, multicentre, phase IV, open-label, superiority clinical trial validates the hypothesis that the administration of BLA is clinically more effective by EI than by II in haematological patients with FN, then the daily routine management of these high-risk patients could be changed to improve their outcomes. TRIAL REGISTRATION: European Clinical Trials Database: EudraCT 2018-001476-37. ClinicalTrials.gov, ID: NCT04233996.

Canine systemic mycosis associated with Chaetomium globosum in a dog.

BACKGROUND: Disseminated mycoses other than aspergillosis are infrequently reported in dogs. CASE REPORT: A 4-year-old female Labrador retriever was evaluated because of hyperthermia, cough and intermittent lameness. Computed tomography showed a soft tissue mass in the cranioventral mediastinum, severe left and central tracheobronchial lymphadenopathy, and moderate bilateral pleural effusion. Magnetic resonance imaging identified an irregular intra-axial well-defined contrast enhancing mass extending from the right frontal lobe to the right thalamus. Fungal culture yielded growth of Chaetomium globosum. CONCLUSION: In this case, the authors report a systemic mycosis in a Labrador retriever caused by C. globosum. To the best of authors' knowledge, this is the first report of systemic disease by this species in veterinary medicine.

Frequency and clinical impact of CDKN2A/ARF/CDKN2B gene deletions as assessed by in-depth genetic analyses in adult T cell acute lymphoblastic leukemia.

Recurrent deletions of the CDKN2A/ARF/CDKN2B genes encoded at chromosome 9p21 have been described in both pediatric and adult acute lymphoblastic leukemia (ALL), but their prognostic value remains controversial, with limited data on adult T-ALL. Here, we investigated the presence of homozygous and heterozygous deletions of the CDKN2A/ARF and CDKN2B genes in 64 adult T-ALL patients enrolled in two consecutive trials from the Spanish PETHEMA group. Alterations in CDKN2A/ARF/CDKN2B were detected in 35/64 patients (55%). Most of them consisted of 9p21 losses involving homozygous deletions of the CDKNA/ARF gene (26/64), as confirmed by single nucleotide polymorphism (SNP) arrays and interphase fluorescence in situ hybridization (iFISH). Deletions involving the CDKN2A/ARF/CDKN2B locus correlated with a higher frequency of cortical T cell phenotype and a better clearance of minimal residual disease (MRD) after induction therapy. Moreover, the combination of an altered copy-number-value (CNV) involving the CDKN2A/ARF/CDKN2B gene locus and undetectable MRD (≤ 0.01%) values allowed the identification of a subset of T-ALL with better overall survival in the absence of hematopoietic stem cell transplantation.

Isolation of Naegleria fowleri from a domestic water tank associated with a fatal encephalitis in a 4 month-old Venezuelan child.

This study describes the association of household water system contamination with the pathogenic Free-Living Amoeba (FLA) Naegleria fowleri and a case of fatal Primary Amoebic Meningoencephalitis (PAM) in a child from the state of Monagas in Venezuela. Amoebae were initially identified by microscopy from a sample of cerebrospinal fluid (CSF) from the child. Direct DNA extraction and specific PCR/sequencing for N. fowleri was also carried out from the same CSF sample. In order to determine a possible environmental source of infection, water samples from the water tank of the child's home and also water bodies recently visited by the child and his family, were examined for the presence of N. fowleri by culture and PCR/sequencing. The results obtained from the collected water samples revealed that only the water tank of the house was positive for N. fowleri. PCR/sequencing showed that the strains isolated from the patient and the water tanks were 100 % identical. Therefore, the house water tank was confirmed as the source of infection in this case, possibly as a result of the occasional immersion of the child´s head under the water while bathing. This case highlights a novel source of thermally polluted water and another threat of N. fowleri infection.

Data on clinical characteristics of a heart failure patients' cohort with reduced ejection fraction and analysis of the circulating values of five different heart failure biomarkers; high sensitivity troponin T, galectin-3, C-terminal propeptide of type I procollagen, soluble AXL and BNP.

In this article, the full description of a heart failure with reduced ejection fraction (HF_REF) cohort of 192 patients is provided. Tables with the baseline demographic, prior history, ECG parameters, echocardiographic parameters, laboratory values and pharmacological treatment of these patients are included. Also, the quartile values of the analyzed circulating biomarkers: high sensitivity Troponin T (hs-TnT), galectin-3 (Gal-3), C-terminal propeptide of type I procollagen (CICP), soluble AXL (sAXL) and Brain Natriuretic Peptide (BNP) are given. The main demographic and clinical features of the patients' subgroups that have hs-TnT, Gal-3, CICP or BNP above the third quartile are described. Tables with Pearson correlation analysis of the HF_REF patients' biomarker levels are included. And Pearson correlation analysis of the HF_REF patients' hs-TnT, Gal-3, CICP levels with patients' biochemical parameters, blood count and inflammation parameters are also described. These data are related to the research articles (AXL receptor tyrosine kinase is increased in patients with heart failure (M. Batlle, P. Recarte-Pelz, E. Roig, M.A. Castel, M. Cardona, M. Farrero, et al., 2014) [1] and Use of serum levels of high sensitivity troponin T, galectin-3 and C-terminal propeptide of type I procollagen at long term follow-up in Heart Failure patients with reduced ejection fraction: comparison with soluble AXL and BNP (M. Batlle, B. Campos, M. Farrero, M. Cardona, B. González, M.A. Castel, et al., 2016) [2].

Use of serum levels of high sensitivity troponin T, galectin-3 and C-terminal propeptide of type I procollagen at long term follow-up in heart failure patients with reduced ejection fraction: Comparison with soluble AXL and BNP.

BACKGROUND: Prognostic biomarkers are needed to improve the management of the heart failure (HF) epidemic, being the brain natriuretic peptides the most valuable. Here we evaluate 3 biomarkers, high sensitivity troponin T (hs-TnT), galectin-3 (Gal-3) and C-terminal propeptide of type I procollagen (CICP), compare them with a recently described new candidate (sAXL), and analyze their relationship with BNP. METHODS: HF patients with reduced ejection fraction (n=192) were included in this prospective observational study, with measurements of candidate biomarkers, functional, clinical and echocardiographic variables. A Cox regression model was used to determine predictors for clinical events, i.e. all-cause mortality and heart transplantation. RESULTS: Hs-TnT circulating values were correlated to clinical characteristics indicative of more advanced HF. When analyzing the event-free survival at a mean follow-up of 3.6years, patients in the higher quartile of either BNP, hs-TnT, CICP and sAXL had increased risk of suffering a clinical event, but not Gal-3. Combination of high sAXL and BNP values had greater predictive value (HR 6.8) than high BNP alone (HR 4.9). In a multivariate Cox regression analysis, BNP, sAXL and NYHA class were independent risk factors for clinical events. CONCLUSIONS: In this HF cohort, hs-TnT is a good HF marker and has a very significant prognostic value. The prognostic value of CICP and sAXL was of less significance. However, hs-TnT did not add predictive value to BNP, while sAXL did. This suggests that elevated troponin has a common origin with BNP, while sAXL could represent an independent pathological mechanism.

[Alimentary and lifestyle changes as a strategy in the prevention of metabolic syndrome and diabetes mellitus type 2: milestones and perspectives]. / Cambios alimentarios y de estilo de vida como estrategia en la prevención del síndrome metabólico y la diabetes mellitus tipo 2: hitos y perspectivas.

A high caloric intake in today's nutrition and a sedentary lifestyle are the main causes of the notable increase in obesity in our society. In turn, this results in an increase in associated pathologies, such as metabolic syndrome and diabetes type 2. In the present work we review most recent studies and programs, which are significant due to their sample size and geographical diversity. It clearly shows that changes in alimentation and lifestyles are an effective instrument for combatting or delaying the onset of these diseases. In this sense, prevention is also key to avoiding serious consequences related to diabetes and metabolic syndrome, which can affect the life of the population.

Diazoxide enhances excitotoxicity-induced neurogenesis and attenuates neurodegeneration in the rat non-neurogenic hippocampus.

Diazoxide, a well-known mitochondrial KATP channel opener with neuroprotective effects, has been proposed for the effective and safe treatment of neuroinflammation. To test whether diazoxide affects the neurogenesis associated with excitotoxicity in brain injury, we induced lesions by injecting excitotoxic N-methyl-d-aspartate (NMDA) into the rat hippocampus and analyzed the effects of a daily oral administration of diazoxide on the induced lesion. Specific glial and neuronal staining showed that NMDA elicited a strong glial reaction associated with progressive neuronal loss in the whole hippocampal formation. Doublecortin immunohistochemistry and bromo-deoxyuridine (BrdU)-NeuN double immunohistochemistry revealed that NMDA also induced cell proliferation and neurogenesis in the lesioned non-neurogenic hippocampus. Furthermore, glial fibrillary acidic protein (GFAP)-positive cells in the injured hippocampus expressed transcription factor Sp8 indicating that the excitotoxic lesion elicited the migration of progenitors from the subventricular zone and/or the reprograming of reactive astrocytes. Diazoxide treatment attenuated the NMDA-induced hippocampal injury in rats, as demonstrated by decreases in the size of the lesion, neuronal loss and microglial reaction. Diazoxide also increased the number of BrdU/NeuN double-stained cells and elevated the number of Sp8-positive cells in the lesioned hippocampus. These results indicate a role for KATP channel activation in regulating excitotoxicity-induced neurogenesis in brain injury.

Robustness of plastic scintillation microspheres in the continuous measurement of different river waters.

Plastic scintillation microspheres (PSm) represent one of the most promising options for monitoring alpha and beta radioactivity in river water. For that reason, a study of the stability of PSm packed into a cell against the continuous flow of river water with different degrees of turbidity was performed over a period of 100h. The results showed that the volume of the cell became stable after 15h of pumping and continued to be stable throughout the 100h of the experiment. During this period of time, the detection efficiency of the PSm, in terms of efficiency*volume, presented mean values of 0.75(3)% for (3)H and 272(11)% for (90)Sr/(90)Y. No dependence on flow time or river water type was observed. The background was also constant for 100h and for the different water types, although (222)Rn should be removed from the water beforehand to prevent its accumulation in the PSm. Since PSm did not present any degradation throughout the whole experiment, PSm can undoubtedly be used for monitoring radioactivity with low reagent consumption, low waste generation and low maintenance costs.