SYSTEMIC AND LOCAL ANTIMICROBIAL ACTIVITY AGAINST PLANKTONIC AND BIOFILM STATE OF MICROORGANISMS IN VITRO AND IN VIVO

Implant-associated infections caused by Candida spp. are difficult to treat due to reduced antimicrobial susceptibility in biofilm. Antifungal susceptibility testing is important due to the increasing number of prosthetic infections caused by Candida spp. The aim of this thesis was to investigate innovative methods using calorimetry for microbial detection and antimicrobial susceptibility testing, as well as treatment activity in validated C. albicans foreign-body infection animal model (part 1). In addition, new materials with intrinsic antimicrobial activity (bioactive glass) were tested (part 2). The first part of the thesis describes a novel real-time method for evaluation of antifungals against yeast, based on measurements of the growth-related heat production by isothermal microcalorimetry. Current methods for evaluation of antifungal agents against yeast have several limitations, especially when combinations of antifungals are investigated. We therefore evaluated the activity of fluconazole, amphotericin B and two echinocandins (caspofungin and anidulafungin) against Candida spp. by microcalorimetry. The minimal heat inhibition concentration (MHIC) was defined as the lowest concentration inhibiting ≥50% (≥90% for amphotericin B) of the heat produced at 24 and 48h for planktonic and biofilm yeast, respectively. Agreement within two-fold dilutions between MHIC and MIC was 50% for fluconazole and 100% for caspofungin, anidulafungin and amphotericin B. As determined by microcalorimetry, echinocandins (especially anidulafungin) were the most active agents against planktonic Candida. Subsequently, antimicrobial treatment strategies for infections caused by Candida albicans in-vivo were investigated. Since C. albicans had not previously been tested in this animal model, we first established an infection profile and investigated the pharmacokinetics of antifungals. In untreated animals, planktonic Candida progressively decreased in cage fluid and was cleared in 8% to 24% of cage fluids, however, Candida biofilm persisted on all cages, i.e. no spontaneous cure of cage- associated infections was observed. In accordance with in vitro experiments, echinocandins showed the highest activity against planktonic C. albicans. Against C. albicans biofilm, caspofungin showed the highest cure rate (25%), whereas cure rates of other antifungals ranged between 8% - 17%, demonstrating the difficulty of eradicating Candida biofilms on implants. The second part of the thesis investigates the activity of bioactive glass (BAG) S53P4 (volumes 1 g and 2 g and sizes 0.5-0.8 mm and <45 µm) against Staphylococcus aureus, S. epidermidis, Escherichia coli, Enterococcus faecalis et Candida albicans by microcalorimetry. BAG is a surface-reactive glass-ceramic biomaterial which is used as implant material to repair and replace diseased or damaged bones. Besides binding chemically to the bone and being osteoconductive, this material has the property to inhibit the biofilm formation. BAG showed good activity against tested mircoorganisms, except for E. faecalis with granula 0.5-0.8 mm. -- Les infections associées aux implants causées par Candida spp. sont difficiles à traiter à cause de la faible susceptibilité du biofilm aux antifongiques. Le test de susceptibilité aux antifongiques est important vu l’augmentation des infections de prothèses dûes à Candida spp. Le but de cette thèse est d’étudier des nouvelles méthodes de détection des microorganismes par calorimétrie et de tester la susceptibilité ainsi que l’activité des antifongiques dans un modèle animal (partie 1). De plus, des nouveaux matériaux avec une activité antimicrobienne intrinsèque (bioactive glass) ont été testés (partie 2). La première partie de la thèse décrit une nouvelle méthode d’évaluation de l’effet des antifongiques en temps réel en se basant sur la chaleur produite lors de la croissance mesurée par microcalorimètre isothermique. Les méthodes actuelles visant à évaluer l’effet des antifongiques sont limitées, surtout lorsqu’il s’agit d’évaluer l’effet des combinaisons d’antifongiques. Nous avons évalué l’activité du fluconazole, de l’amphotericin B et des echinocandines (caspofungine et anidulafungine), sur différentes souches de Candida spp. La concentration minimale d’inhibition de chaleur (CMIC) a été définie comme étant la plus petite concentration inhibant ≥50% (≥90% pour l’amphotericine B) de la chaleur produite à 24h et 48h pour la croissance planctonique et du biofilm. La concordance entre CMIC et la concentration minimale d’inhibition (CMI), avec 2 dilutions de marge, était de 50% pour fluconazole et 100% pour les echinocandines et l’amphotericin B. Comme déterminé par microcalorimétrie, les echinocandines (surtout l’anidulafungine) ont montré une meilleure activité contre la croissance planctonique de Candida. Nous avons ensuite étudié les traitements antimicrobiens contre C. albicans in vivo. Étant donné l’absence d’études avec ce modèle animal avec C. albicans, nous avons d’abord établi un profil d’infection et étudié la pharmacocinétique des antifongiques. Chez les animaux non traités, Candida planctonique a montré une décroissance progressive dans le fluide des cages, tout en restant présent sous forme de biofilm. Pas de cure spontanée des cages infectées a été observée. En accord avec les expériences in vitro, les echinocandines ont montré une meilleure activité contre C. albicans planctonique. Contre le biofilm la caspofongine montre le plus haut taux de guérison (25%), contrairement aux autres antifongiques où le taux de guérison allait de 8% à 17%, démontrant ainsi la difficulté rencontrée dans l’éradication du biofilm à C. albicans. La deuxième partie étudie l’activité du bioactive glass (BAG) S53P4 (volumes 1 and 2 g et diamètres de 0.5-0.8 mm et <45 µm) contre Staphylococcus aureus, S. epidermidis, Escherichia coli, Enterococcus faecalis et C. albicans. BAG est un biomatériel en vitre-céramique à surface réactive utilisé en tant qu’implant pour réparer et remplacer les os endommagés ou fracturés. En plus d’avoir la capacité de se lier chimiquement à l’os et être ostéoconductif, ce matériel a la caractéristique d’inhiber la formation du biofilm. BAG a montré une bonne activité contre les microorganismes testés à l’exception de E. faecalis avec les granules de 0.5-0.8 mm de diamètre

Composite Gels Based on Poly (Vinyl alcohol) for Biomedical Uses

Nowadays, poly (vinyl alcohol) (PVA) hydrogels are being studied for several biomedical applications such as joint replacement, wound dressings and controlled drug-releasing devices, among others. Reinforced PVA hydrogels show good mechanical properties and are a suitable option to replace cartilages. Furthermore, these materials can prevent loss of body fluids, be a barrier against bacteria and also permeable to oxygen, for these all interesting properties, they are used like wound dressings. For drug delivery systems a material that can control the dose and release at the site of action is desirable, this can be accomplished using hydrogels, which are loaded with a drug, and then they can release it when an external stimulus (light, temperature, magnetic field, etc.) takes place. The aim of this work was to obtain composite hydrogels for the previously mentioned biomedical applications. Hydroxyapatite (HA) reinforced PVA gels were prepared for potential uses as cartilage replacement, HA improves the mechanical, tribological and fixing properties of the polymer, reaching values similar to that of the cartilages. For wound dressings, the hydrogel was reinforced with bentonite (clay) in order to increase the dimensional stability and antimicrobial properties. Gels with controlled drug release capability under magnetic stimulation (ferrogels) were also synthesized and characterized here.Fil: Hoppe, Cristina Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación en Ciencia y Tecnología de Materiales (i); ArgentinaFil: Alvarez, Vera Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación en Ciencia y Tecnología de Materiales (i); ArgentinaFil: Maiolo, Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación en Ciencia y Tecnología de Materiales (i); ArgentinaFil: Gonzalez, Jimena Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación en Ciencia y Tecnología de Materiales (i); Argentin

Thermogenic diagnosis of periprosthetic joint infection by microcalorimetry of synovial fluid

Background: Synovial fluid culture is the standard investigation for the preoperative diagnosis of periprosthetic joint infection (PJI). However, the culture has limited sensitivity and requires several days until result. We evaluated the value of isothermal microcalorimetry for real-time diagnosis of PJI based on heat produced by microbial growth in synovial fluid. Methods: Patients undergoing aspiration of prosthetic hip or knee joint before revision surgery were prospectively included between 2014 and 2015. The performance of microcalorimetry was compared to synovial fluid culture using McNemar’s chi-squared test. Pearson’s correlation coefficient was calculated for synovial fluid leukocyte count and microcalorimetric heat. Results: Of 107 included patients (58 knee and 49 hip prosthesis), PJI was diagnosed in 46 patients (43%) and aseptic failure in 61 patients (57%) according to institutional criteria. In 26 PJI cases (56%) the pathogen grew in synovial fluid and intra-operative cultures. The sensitivity of synovial fluid culture and microcalorimetry was both 39% and the results were concordant in 98 patients (92%). In patients with PJI, microcalorimetry missed 4 pathogens which grew in synovial fluid culture, whereas culture missed 4 pathogens detected by microcalorimetry. A linear correlation (r = 0.366) was found between leukocyte count and microcalorimetric heat in synovial fluid (p < 0.001). The median time to positivity of microcalorimetry was 9 h (range, 1–64 h) vs. 3 days for cultures (range, 1–14 days). Conclusion: Microcalorimetry of synovial fluid allows thermogenic diagnosis of periprosthetic joint infection in synovial fluid. The diagnostic performance of synovial fluid microcalorimetry is comparable to culture and delivers results considerably faster. Trial registration: This prospective study was registered on August 21, 2015 with the public clinical trial identification NCT02530229

Epstein-Barr Virus (EBV)-Associated Haemophagocytic Syndrome

We describe the case of a 17- year old female who developed fatal haemophagocytic syndrome (HPS) one month following acute infection caused by Epstein-Barr virus (EBV). Despite initiation of treatment and reduction of EBV load, laboratory signs of HPS as severe cytopenia, hypofibrinogenemia, hyperferritinemia and hypertriglyceridemia persisted, and the patient died of multiorgan failure. HPS is a rare, but life-threatening complication of EBV infection

In vitro evaluation of structural factors favouring bacterial adhesion on orthodontic adhesive resins

Bacterial adhesion to the surface of orthodontic materials is an important step in the formation and proliferation of plaque bacteria, which is responsible for enamel demineralization and periodontium pathologies. With the intent of investigating if adhesive resins used for bracket bonding are prone to bacteria colonization, the surface roughness of these materials has been analyzed, combining information with a novel methodology to observe the internal structures of orthodontic composites. Scanning electron microscopy, combined with focus ion bean micromachining and stylus profilometry analyses, were performed to evaluate the compositional factors that can influence specific pivotal properties facilitating the adhesion of bacteria to the surface, such as surface roughness and robustness of three orthodontic adhesive composite resins. To confirm these findings, contact angle measurements and bacteria incubation on resin slide have been performed, evaluating similarities and differences in the final achievement. In particular, the morphological features that determine an increase in the resins surface wettability and influence the bacterial adhesion are the subject of speculation. Finally, the focused ion beam technique has been proposed as a valuable tool to combine information coming from surface roughness with specific the internal structures of the polymers

Venous Thromboembolism in Lymphoma: Risk Stratification and Antithrombotic Prophylaxis

Lymphoma is listed among the neoplasias with a high risk of venous thromboembolism (VTE). Risk factors for VTE appear to differ from risk factors in solid tumors. We review the literature of the last 20 years for reports identifying these risk factors in cohorts consisting exclusively of lymphoma patients. We selected 25 publications. The most frequent studies were analyses of retrospective single-center cohorts. We also included two reports of pooled analyses of clinical trials, two meta-analyses, two analyses of patient registries, and three analyses of population-based databases. The VTE risk is the highest upfront during the first two months after lymphoma diagnosis and decreases over time. This upfront risk may be related to tumor burden and the start of chemotherapy as contributing factors. Factors consistently reported as VTE risk factors are aggressive histology, a performance status ECOG 65 2 leading to increased immobility, more extensive disease, and localization to particular sites, such as central nervous system (CNS) and mediastinal mass. Association between laboratory values that are part of risk assessment models in solid tumors and VTE risk in lymphomas are very inconsistent. Recently, VTE risk scores for lymphoma were developed that need further validation, before they can be used for risk stratification and primary prophylaxis. Knowledge of VTE risk factors in lymphomas may help in the evaluation of the individual risk-benefit ratio of prophylaxis and help to design prospective studies on primary prophylaxis in lymphoma

Hematopoiesis and immune reconstitution after CD19 directed chimeric antigen receptor T‐cells (CAR‐T): A comprehensive review on incidence, risk factors and current management

Impaired function of hematopoiesis after treatment with chimeric antigen T-cells (CAR-T) is a frequent finding and can interest a wide range of patients, regardless of age and underlying disease. Trilinear cytopenias, as well as hypogammaglobulinemia, B-cell aplasia, and T-cell impairment, can severely affect the infectious risk of CAR-T recipients, as well as their quality of life. In this review, we provide an overview of defects in hematopoiesis after CAR-T, starting with a summary of different definitions and thresholds. We then move to summarize the main pathogenetic mechanisms of cytopenias, and we offer insight into cytomorphological aspects, the role of clonal hematopoiesis, and the risk of secondary myeloid malignancies. Subsequently, we expose the major findings and reports on T-cell and B-cell quantitative and functional impairment after CAR-T. Finally, we provide an overview of current recommendations and leading experiences regarding the management of cytopenias and defective B- and T-cell function

The serotonergic central nervous system of the Drosophila larva: anatomy and behavioral function.

The Drosophila larva has turned into a particularly simple model system for studying the neuronal basis of innate behaviors and higher brain functions. Neuronal networks involved in olfaction, gustation, vision and learning and memory have been described during the last decade, often up to the single-cell level. Thus, most of these sensory networks are substantially defined, from the sensory level up to third-order neurons. This is especially true for the olfactory system of the larva. Given the wealth of genetic tools in Drosophila it is now possible to address the question how modulatory systems interfere with sensory systems and affect learning and memory. Here we focus on the serotonergic system that was shown to be involved in mammalian and insect sensory perception as well as learning and memory. Larval studies suggested that the serotonergic system is involved in the modulation of olfaction, feeding, vision and heart rate regulation. In a dual anatomical and behavioral approach we describe the basic anatomy of the larval serotonergic system, down to the single-cell level. In parallel, by expressing apoptosis-inducing genes during embryonic and larval development, we ablate most of the serotonergic neurons within the larval central nervous system. When testing these animals for naïve odor, sugar, salt and light perception, no profound phenotype was detectable; even appetitive and aversive learning was normal. Our results provide the first comprehensive description of the neuronal network of the larval serotonergic system. Moreover, they suggest that serotonin per se is not necessary for any of the behaviors tested. However, our data do not exclude that this system may modulate or fine-tune a wide set of behaviors, similar to its reported function in other insect species or in mammals. Based on our observations and the availability of a wide variety of genetic tools, this issue can now be addressed