The Detection Limits of Legionella According to the EU Directive 2020/2184. Could That Be Too Permissive?

INTRODUCTION: The problem of detecting legionella after a case of legionellosis from the source of environmental contamination has been known since a long time ago. Legionella is a bacterium present in various natural and artificial habitats and especially in surface fresh waters. It is found in greater concentration in warm waters, at temperatures between 20 °C and 42 °C. The greatest risk factor for humans is represented by the presence of Legionella in water distribution systems in hospitals, medical equipment (e.g. respirators, dialyzers, inhalers, humidifiers, water, massage equipment used in balneotherapy) and turbines used in dental practices, especially for hospitalized individuals. In the EU directive 2020/2184, issued by the European parliament on 16/12/2020, the concentration of Legionella was added to the parameters to be determined in assessing the quality of drinking water intended for human consumption. The objectives were to improve the quality standard of drinking water, reduce the consumption of bottled water and consequently reduce plastic waste. The WHO notes that Legionella causes the greatest burden from a health point of view and it is included among the parameters that require careful monitoring with a limit of less than 1000 CFU/L. The aim of this report was to evaluate the new EU directive 2020/2184 on the light of our laboratory experience. MATERIALS AND METHODS: A total of 459 samples were processed at our Hygiene of food Laboratory - Department of Medical Sciences and Public Health. All statistical analyses were conducted using the SPSS statistical package (version 23 for Windows. SPSS, Inc. Chicago, Ill). RESULTS AND DISCUSSION: Of the 67 structures examined where the cases occurred, 35 showed samples with at least one over-threshold value considering the reference value of 100 CFU/L, whereas using the new limit of 1000 CFU/L, only 25 structures resulted as having at least one sample above the threshold. In our experience as a regional reference laboratory for Legionella research, the increase from 100 CFU/L to 1,000 CFU/L could lead to a lower alert level. In fact, in the period between October 2017 and October 2021, the median value of CFU/L in presence of a case was 0 (0-100). Despite the large amount of studies on Legionella only a few relate the withdrawals and the consequent CFU/L with the confirmed cases of legionellosis, as in our analysis. The 75° percentile values of the Legionella concentration equal to 100 CFU/L in all samples associated with cases and clusters leads us to hypothesize that the limit equal to 1000 CFU/L that will be introduced for environmental monitoring as per recent European regulations may not be sufficiently protective for minimizing risk in the population, especially in healthcare facilities where fragile patients are assisted.

A numerical study towards shape memory alloys application in orthotic management of pediatric knee lateral deviations.

Exerting a constant load would likely improve orthosis effectiveness in treating knee lateral deviations during childhood and early adolescence. Shape memory alloys are potential candidates for such applications due to their so called pseudoelastic effect. The present study aims to quantitatively define the applicable mechanical loads, in order to reduce treatment duration while avoiding tissular damage and patient discomfort. This is essential for performing a more efficient design of correction devices. We use a patient-specific finite elements model of a pediatric knee to determine safe loading levels. The achievable correction rates are estimated using a stochastic three-dimensional growth model. Results are compared against those obtained for a mechanical stimulus decreasing in proportion to the achieved correction, emulating the behavior of conventional orthoses. A constant flexor moment of 1.1 Nm is estimated to change femorotibial angle at a rate of (7.4 ± 4.6) deg/year (mean ± std). This rate is similar to the achieved by more invasive growth modulation methods, and represents an improvement in the order of 25% in the necessary time for reducing deformities of (10 ± 5) deg by half, as compared with conventional orthoses.

Trends of Antimicrobial Consumption in Hospital: Tackling the Hidden Part of the Iceberg with an Electronic Personalised Prescription Software for Antimicrobial Stewardship.

BACKGROUND: The monitoring of antibiotic prescriptions is of fundamental importance in the hospital setting. Inappropriate prescriptions could cause an unjustified exposure of patients to the risk of ADR (adverse drug reactions) and increase the risk of spreading the ecological resistance of hospital microorganisms. The use of IT media is essential in antimicrobial stewardship programs. OBJECTIVE: The purpose of this work was to evaluate the variation in the exposure index to antibiotics following the adoption of electronic pharmacist-controlled prescriptions in 2015. METHODS: Electronic Personalised Prescription Software (EPPS) was introduced in our University Hospital in 2015. The exposure index to antibiotics was expressed per WHO methodology in DDD (defined daily dose)/100 patient days (DPD). The changes in DPDs over the 2015-2020 period were calculated as percentages and through linear regressions. The analysis was performed using SPSS® (IBM). RESULTS: Following the introduction of EPPS, there was a progressive decline in DPDs during the 2015-2020 period from 98.9 to 65.1 (R2 = 0.687, p = 0.041). This could mainly be linked to the decreased use of ATC class J01CR - penicillin association, including beta-lactamase inhibitors (DPD 2015 39.9; DPD 2020 11.5; variation -71.1%). Expenditure progressively decreased from € 427,000 in 2015 to € 269,000 in 2020. CONCLUSION: The use of EPPS was shown to be useful for pharmacists in implementing proper antibiotic dispensing practices; the avoidance of inappropriate prescriptions leads to a better monitoring of DPDs and the related expenditure which is the main goal of antibiotic stewardship programs.

A tool for solving bone growth related problems using finite elements adaptive meshes.

Long bones geometry changes in response to longitudinal growth in the epiphyseal plates and hydroxyapatite apposition in the periosteum. Due to its relevance for growth modulation and orthotics performance, researchers have extensively modeled these phenomena, using the finite elements method for it almost since the introduction of modern computers. This is a rather complex task that, besides the inherent difficulty of solving the models equations, requires considering a moving boundary. Here, the development of a new computational tool for its resolution is described. A generalized formulation of these problems is established based on the most common approaches taken in the literature and a novel finite elements algorithm is proposed for its resolution. The later allows a significant reduction of the spatial discretization requirements, the computational cost and the numerical errors associated with more classical approaches. The potentiality of the method is demonstrated by its application to three cases of practical interest, namely, hemiepiphysiodesis treatment, growth in the distal femur and bone remodeling around hip prosthesis. Eight relevant cases of study and an open source implementation of the proposed algorithm are also provided as supplementary material.

Mechanobiological based long bone growth model for the design of limb deformities correction devices.

A mechanobiological model of bone growth aimed for the design of medical devices for the treatment of limb deformities during childhood and adolescence was developed. Dimensional analysis was introduced as a tool for the systematic evaluation of the influence attributed to different factors that might modify the bone growth process. Simplifications were proposed, allowing the reduction of bone growth relevant parameters to four non-dimensional numbers, representing the chondrocyte sensitivity to stress, the epiphyseal plate geometry, the bone rigidity and the time. Benchmark situations considered for model validation were bone growth under normal conditions and an epiphyseal stapling treatment. A finite elements approach was used to analyze bone growth in the distal portion of the femur. Results are shown to be consistent with corresponding clinical data published in the literature, which indicates the potential of the here proposed method for the design of specific devices and treatments.

Platelet composition and function in patients undergoing cardiopulmonary bypass for heart surgery.

BACKGROUND: Previous studies showed severe biochemical and functional damage to platelets in patients undergoing cardiopulmonary bypass for cardiac surgery, and suggested that this derived from the proteolytic action of plasmin on the platelet surface. METHODS: A double-blind study was carried out to compare platelet function and composition in patients randomized to receive the protease inhibitor aprotinin or placebo during reoperation for valvular prosthesis replacement or coronary artery bypass grafting. RESULTS: Flow cytometry with specific monoclonal antibodies and polyacrylamide gel electrophoresis did not show any significant proteolysis of platelet glycoprotein Ib and IIb-IIIa either in the placebo or the aprotinin group. Functional studies were consistent with these results, since ristocetin-induced platelet agglutination was unchanged and platelet aggregation and ATP release induced by collagen and ADP were only slightly reduced by cardiopulmonary bypass. These mild defects in platelet function were partially prevented by aprotinin infusion. CONCLUSIONS: On the basis of our data and those from literature, we suggest that platelets may be affected very little or severely damaged during cardiopulmonary bypass for cardiac surgery, probably depending on some aspects of the technical procedure which remain to be identified. Aprotinin infusion significantly protects platelets in the latter condition, while its role is obviously slight in the former.

Ristocetin-induced platelet agglutination stimulates GPIIb/IIIa-dependent calcium influx.

We found that intracellular Ca2+ concentration ([Ca2+]i) increased during ristocetin-induced agglutination of aequorin loaded platelets resuspended in plasma. Chelation of extracellular Ca2+ had no effect on platelet clumping, but delayed and greatly reduced Ca2+ increase, indicating that it derived for the most part from Ca2+ influx. Nine monoclonal antibodies (MA) against glycoprotein (GP) Ib largely prevented ristocetin-induced platelet clumping and [Ca2+]i increase, while three anti-GPIb MA with no effect on platelet clumping did not interfere with Ca2+ movement. In unstirred samples platelet agglutination was greatly reduced and [Ca2+]i increase was abolished, suggesting that close platelet-to-platelet contact, in addition to von Willebrand factor (vWF) binding to GPIb, is necessary for Ca2+ transient. Nine MA against GPIIb/IIIa, the gly-arg-gly-asp-ser (GRGDS) peptide and GPIIb/IIIa complex dissociation had no effect on platelet agglutination, but significantly reduced Ca2+ increase. Our results suggest that platelet clumping induced by vWF binding to GPIb is responsible for GPIIb-IIIa dependent Ca2+ influx.

Acquired cyclic thrombocytopenia-thrombocytosis with periodic defect of platelet function.

A periodic fall of platelet number characterizes an acquired pathological condition named cyclic thrombocytopenia. We observed a patient in whom the episodes of thrombocytopenia (platelet number less than 50 x 10(9)/l) were followed regularly by thrombocytosis (700-2300 x 10(9) platelets/l). The period of platelet count fluctuation was about 40 d. Morphological examination of bone marrow showed the cyclic disappearance of mature and immature megakaryocytes; bone marrow cultures revealed a periodic severe defect of both multilineage and single-lineage progenitor cell growth. When platelet count was falling, a mild defect of platelet aggregation and ATP release was observed, while platelet function was normal when platelet count was rising. Prednisone, thymopentin, high-dose intravenous gamma-globulin and splenectomy were without effect. After 4 years of cyclic platelet and megakaryocyte fluctuations, stable amegakaryocytic thrombocytopenia developed and the patient died of haemorrhagic stroke.

Heparin infusion facilitates ex vivo spontaneous platelet aggregation in patients with acute myocardial infarction who have undergone thrombolytic therapy.

We investigated ex vivo spontaneous platelet aggregation (SPA) in platelet-rich plasma in 37 patients with acute myocardial infarction. It occurred in about 50% of subjects receiving heparin after streptokinase treatment, while it rarely took place in patients who did not receive either streptokinase or heparin and in those treated with streptokinase alone. The study of patients receiving heparin for deep vein thrombosis suggested that SPA may derived from adenosine diphosphate released from platelets during sample handling. We suggest that heparin infusion may facilitate ex vivo platelet activation and that this mechanism is operative in patients with acute myocardial infarction who have undergone thrombolytic therapy.

Cryopreservation of human platelets using dimethyl sulfoxide and glycerol-glucose: effects on "in vitro" platelet function.

BACKGROUND: The technique of freezing blood platelets could be very useful in the transfusion support of thrombocytopenic patients. The best method of platelet cryopreservation still remains an object of debate, though it has been suggested that dimethyl sulfoxide (DMSO) is more effective than glycerol-glucose as a cryopreservative. However, few studies have directly compared platelets cryopreserved with different methods. METHODS: We compared "in vitro" function of platelets cryopreserved with 5% dimethyl sulfoxide (DMSO) or 3% glycerol-glucose at -140 degrees C. Platelet aggregation and release reaction were studied with a Lumi aggregometer, thromboxane B2 (TxB2) production by radioimmunoassay, and Ca++ movement by the Fura 2 method. RESULTS: Cryopreservation with both of the methods dramatically reduced the ability of platelets to release ATP and to aggregate in response to single agonists. In contrast, cryopreserved platelets maintained their ability to aggregate after stimulation with paired agonists and to produce TxB2. Cytoplasmic Ca++ increase induced by thrombin was observed in the glycerol-preserved platelets, while it was nearly absent in the DMSO-preserved ones. CONCLUSIONS: We suggest that cryopreservation with glycerol-glucose or DMSO induces similar defects of platelet function. The damage is severe, but platelets are still able to respond to strong stimulation.

Defect of platelet aggregation and adhesion induced by autoantibodies against platelet glycoprotein IIIa.

A young patient developed chronic idiopathic thrombocytopenic purpura. Prednisone therapy normalized platelet number, but bleeding symptoms did not disappear. Platelet function was severely impaired, since platelet aggregation, ATP release and adhesion to collagen and subendothelial matrix were significantly reduced. Plasma and purified immunoglobulins of the patient reproduced the functional defects in normal platelets. Immunoblotting revealed that patient's plasma contained an antibody reacting with a component of platelets with the same electrophoretic mobility of glycoproteins IIIa of normal platelets. Moreover, patient's plasma inhibited the binding of an anti-GPIIb/IIIa monoclonal antibody to platelet surface. Additional immunosuppressive therapy with prednisone and azathioprine normalized platelet function and induced the disappearance of bleeding symptoms.

Platelet function after in vivo and in vitro treatment with thrombolytic agents.

Whereas in vitro studies showed that plasmin may induce both inhibition and activation of platelets, in vivo and ex vivo investigations suggested that thrombolytic agents are responsible for platelet stimulation. To gain further information on this topic, ex vivo platelet function was studied in 24 subjects with acute myocardial infarction treated with streptokinase or recombinant tissue-type plasminogen activator (rt-PA). Ten patients with acute myocardial infarction who did not receive thrombolytic treatment were also investigated. The data shows that at the end of thrombolytic infusion, the maximal extent of platelet aggregation and adenosine triphosphate release was reduced in treated patients compared with that in untreated ones. In subjects treated with streptokinase, the defect in platelet aggregation derived from both cellular and plasmatic defects. Plasmatic beta-thromboglobulin concentration was significantly reduced after streptokinase, but unchanged after rt-PA. Three days after thrombolytic treatment, platelet aggregation of patients receiving streptokinase or rt-PA was not significantly different from that of untreated subjects. A similar defect in platelet function was obtained in vitro, incubating normal platelet-rich plasma with pharmacologic concentrations of streptokinase. Again, platelet function defect derived from both cellular and plasmatic damages. It cannot be excluded that platelet activation occurs in patients with acute myocardial infarction during the very early phases of thrombolytic treatment. However, it is suggested that a transient defect in platelet function follows both streptokinase and rt-PA infusion.

Effect of anagrelide on platelet count and function in patients with thrombocytosis and myeloproliferative disorders.

BACKGROUND: Anagrelide is a quinazolin compound developed initially as an inhibitor of platelet aggregation. Since "in vivo" studies demonstrated that it was responsible for thrombocytopenia in humans, anagrelide has been used recently in a small number of patients with thrombocytosis and myeloproliferative disorders. Platelet count was well controlled in the large majority of patients, and only minimal side effects were observed. PATIENTS: Eight patients (5 with essential thrombocythemia, 2 with chronic granulocytic leukemia, and 1 with idiopathic myelofibrosis) received anagrelide (induction dose 4 mg/die; mean maintenance dose 2 mg/die; mean observation time 26 weeks). Complete blood counts were determined 4 times during the first month, and subsequently every month. "In vivo" and "ex vivo" platelet function was studied before anagrelide and after 4 and 10 days of therapy. RESULTS: Platelet count was reduced and maintained below 500 x 10(9)/L in 5 of 8 patients. Headache, palpitation/tachycardia, gastrointestinal symptoms and a decrease in hemoglobin were the side effects. Anagrelide did not modify the leukocyte count or "in vivo"/"ex vivo" platelet function. CONCLUSIONS: Anagrelide may control thrombocytosis in patients with myeloproliferative disorders, even when traditional drugs have failed. When required, anti-aggregating drugs may be associated with anagrelide, since it has no effect on platelet function.

Aggregation of human platelets stimulates calcium ion movement and release reaction.

We studied the effect of inhibition of platelet aggregation (obtained by omitting sample stirring and by the addition of ASA and GRGDS peptide) on ATP secretion and Ca(2+) movements. When collagen was the agonist, platelet aggregation stimulated release reaction and Ca(2+) movements in both the presence and absence of extracellular Ca(2+). When platelets were stimulated by thrombin, ATP release and Ca(2+) movements were largely independent of aggregation. Our data suggest that platelet aggregation stimulates Ca(2+) movement, and that this phenomenon of feedback amplification of platelet activation plays an important role in platelet function when collagen is the agonist, while it has little or no role when thrombin is the stimulus.

Ultrasonography and blood flow velocity waveform assessment in human fetuses.

The authors report their experience with the use of ultrasonography and fetal blood flow measurement in the evaluation of fetal conditions recording patients with previous repeated immunologic abortion (R.I.A.) and with pre-eclampsia. In both groups of patients, the two biophysical methods, proved to be extremely valuable indicators of the fetal conditions.