IMPLEMENTACIÓN DE LA SIMULACIÓN IN SITU EN UN HOSPITAL PEDIÁTRICO DE ALTA COMPLEJIDAD DURANTE LA PANDEMIA COVID-19 / IMPLEMENTATION OF THE IN-SITU SIMULATION IN A HOSPITAL DISCHARGE PEDIATRIC COMPLEXITY DURING THE COVID-19 PANDEMIC

Introducción: El aprendizaje basado en simulación se ha usado ampliamente para mejorar la respuesta de los integrantes del equipo de salud ante situaciones de crisis. La pandemia por COVID-19 planteó el desafío de utilizar la simulación in situ como estrategia de capacitación. Objetivo: Describir el impacto del entrenamiento en RCP en pacientes COVID-19 mediante simulación in situ según la autopercepción del equipo de salud participante y comparar los resultados entre aquellos que hicieron un taller previo de manejo de vía aérea y RCP en pacientes con SARS-CoV -2 y los que no lo realizaron. Material y Método: Estudio exploratorio-descriptivo, desde marzo a junio de 2020, por medio de un cuestionario anónimo estandarizado a los participantes luego de 30 escenarios de simulación in situ. Las variables cualitativas se registraron con valores del 0 al 5 donde 1: nada; 2: bajo; 3: medio; 4: alto y 5:máximo. Resultados: De los 55 participantes, el 38 % (n=21) habían realizado previamente un taller de manejo de vía aérea avanzada y RCP en paciente COVID-19. El 40,7% expresó que disminuyó el temor a la asistencia de un paciente sospechoso o positivo en un nivel máximo (31,5% nivel alto) 81,5 % manifestó que le sirvió en grado máximo para reconocer la importancia del trabajo en equipo y designación de roles (13% grado alto). Para el 65% tuvo una utilidad máxima (18,5% alta) en adecuación del carro de paro y elaboración de un kit específico. Se halló diferencia significativa en la disminución del temor a la asistencia al comparar el nivel de respuesta entre los que habían realizado un taller previo versus los que no ( p= 0,013) Conclusión: La simulación in situ resultó ser una herramienta útil que ayudó en gran medida a disminuir el temor, mejorar habilidades comunicacionales y el trabajo en equipo Los resultados obtenidos fueron aún mejores cuando el escenario fue complementado con un taller de manejo de vía aérea avanzada y RCP

Introduction: Simulation-based learning has been widely used to improve the response of health team members to crisis situations. The COVID-19 pandemic posed the challenge of using on-site simulation as a training strategy.Objective: Analyze the impact of CPR training in COVID-19 patients in situ simulation according to the self-perception of the health team and compare the results between those who did a previous workshop and those who did not. Material and Method: Exploratory-descriptive study, from March to June 2020, through a standardized anonymous questionnaire to the participants after 30 simulation scenarios in situ. The qualitative variables were registered with values from 0 to 5 where 1: nothing; 2: low; 3: medium; 4: high and 5: maximum. Results: Of the 55 participants, 21 (38%) have previously conducted a workshop on advanced airway management and CPR in a CO- VID-19 patient. 40,7% expressed that the fear of attending a suspicious or positive patient decreased at a maximum level (31,5% high level). 81,5% stated that it served them to a maximum degree to recognize the importance of teamwork and role designation (13% high degree). For 65% it had a maximum utility (18.5% high) in adapting the stop car and making a specific kit. When we compared the subgroup that carried out the previous workshop with the one that did not, differences were found in most of the variables, highlighting the decrease in fear. Conclusion: The in situ simulation turned out to be a useful tool that greatly helped reduce fear and improve communication skills and teamwork. The results were obtained even better when the scenario was complemented with an advanced airway management and CPR workshop

Matrix metalloproteinase (MMP)-9: A realiable marker for inflammation in early human trichinellosis.

Matrix Metalloproteinases (MMPs) are involved in many physiological and pathological processes. As regards parasitic infections, the role of these proteins has been particularly studied in malaria, neurocysticercosis and angiostrongyloidosis. Recently, we evaluated serum levels of MMP-9 and -2 (gelatinases) in mice experimentally infected with Trichinella spiralis or Trichinella pseudospiralis, which cause different degrees of myositis and we found their significant increase in the former and, at a lesser extent, in the latter, thus suggesting the possibility that these gelatinases, particularly MMP-9, represent a marker of inflammation. Our aim was to evaluate the levels of MMP-9 and 2 in trichinellosis patients, to assess their possible clinical significance. Serum samples from 31 Trichinella britovi-infected individuals (20 males and 11 females), living in Tuscany, Central Italy, were analysed for MMP-9 and MMP-2 serum levels. Patients acquired infection with Trichinella after consuming raw or undercooked meat of wild boar. Their median age was 49±0.33years (range from 7 to 91). Sera was collected before starting anti-inflammatory treatment, aliquoted and stored at -20°C until use. Sera from healthy subjects was considered as controls. The gelatinolytic activity of MMPs was analysed by gelatin zymography on 8% polyacrylamide-SDS gels containing 0.1% porcine gelatin, under non-reducing conditions. Clear bands corresponding to the digested areas were evaluated with an appropriate software. MMP-9 levels were additionally determined in 15 patients using a commercial ELISA kit for human MMP-9. The zymographic analysis of the gels showed the presence in serum samples of gelatinase bands at approximately 125-kDa, 92-kDa and 72-kDa, corresponding to the MMP-9/Neutrophil gelatinase-associated lipocalin (NGAL) complex and proenzyme forms of MMP-9 and MMP-2, respectively. A significant (p<0.01) increase in gelatinolytic activity in patients compared to the control group was observed for pro-MMP-9 in 25 out of 31. The mean increase in activity was 39.25%±16.67%. No significant differences were observed for pro-MMP-2 activity. The MMP-9 levels detected by ELISA showed significant correlation with zymographic data (r2=0.62, p<0.003) and were higher in more affected patients (suffering diarrhea, facial edemas and myalgia). In conclusion, MMP-9 might be considered as a marker of inflammation in T. britovi patients. On the contrary, MMP-2 did not result significantly different in patients, compared to controls.

Seroprevalence for toxoplasmosis in individuals living in north west Tuscany: access to Toxo-test in central Italy.

This survey aimed to estimate the prevalence of anti-Toxoplasma IgG and IgM antibodies in people living in north west Tuscany (central Italy) and to investigate the adherence to antenatal screening programs and access to the Toxo-test as well. Sera from a large sample of individuals suspected to have acute infection or from pregnant women (10,352 subjects) aged between 1 day and over 70 years were analysed for both IgG and IgM anti-Toxoplasma antibodies using an immunoenzymatic method or a chemo-luminescent immunoassay. Overall, the seroprevalence of IgG antibodies was 21.4% (95% CI 20.62-22.20). A positive trend according to age was found, with low positivity observed in younger age groups. Among women of reproductive age the prevalence of IgG antibodies was 19.4% (95% CI 18.64-20.26). The overall IgM seroprevalence was 1.07% (95% CI 0.87-1.27). A low IgM prevalence was also observed in women of reproductive age (0.8%; 95% CI 0.65-1.03). Our study seems to indicate that primary prevention is widespread among women. However, an epidemiological surveillance system for toxoplasmosis should be implemented, to assess the risk of congenital toxoplasmosis and to determine the true burden of disease in adults.

Rapid identification and antimicrobial susceptibility profiling of Gram-positive cocci in blood cultures with the Vitek 2 system.

Rapid identification and antimicrobial susceptibility profiling of the bacteria in blood cultures can result in clinical and financial benefits. Addition of saponin to the fluid from blood culture bottles promotes the recovery of the bacteria and thus may shorten the turnaround time of the microbiological analyses. In this study we compared the identification and susceptibility profiles of saponin-treated and untreated (standard method) blood cultures monomicrobial for Gram-positive cocci using Vitek 2. We concordantly identified 49 (89%) of 55 monobacterial cultures using the results with the standard method as reference. Complete categorical agreement between the susceptibility profiles with the new and the standard method was found for 26 (53%) of 49 isolates, while discrepancies were seen for 23 (47%) cultures. E-tests indicated that the new method resulted in a correct susceptibility profile for 8 (35%) of these 23 blood cultures. Therefore, 34 (69%) of 49 cultures showed a concordant/correct susceptibility profile for all antimicrobials with an overall error rate of 2.3%. Thus, addition of saponin to the fluid from blood culture bottles of the Bactec 9240 leads to the rapid (results available >or=12 hours earlier) and reliable identification and susceptibility profiling of Gram-positive cocci in blood cultures with Vitek 2.

Rapid identification and antimicrobial susceptibility testing of Gram-positive cocci in blood cultures by direct inoculation into the BD Phoenix system.

Rapid identification and antimicrobial susceptibility testing (AST) of the causative agent(s) of bloodstream infections are essential for the selection of appropriate antimicrobial therapy. To speed up the identification and AST of the causative agent, the fluid from blood culture bottles of a Bactec 9240 instrument (Becton Dickinson) containing Gram-positive cocci was mixed with saponin. After a 15-min incubation, the bacteria were harvested and transferred to the appropriate panel of a BD Phoenix automated microbiology system (Becton Dickinson) for identification and AST. With this approach (referred to as the direct method), we concordantly/correctly identified 56 (82%) of 68 monomicrobial cultures using the results obtained with the method currently used in our laboratory (current method) as comparator. Two (3%) isolates could not be identified and ten (15%) were misidentified. Complete agreement, concerning clinical susceptibility categories and MIC values, between the AST results determined with the direct method and the current method was found for 32 (55%) of 58 isolates. The E-test indicated that the direct method yielded a correct susceptibility profile for 13 of the remaining 26 blood culture isolates. Therefore, a concordant/correct susceptibility profile (with all antimicrobial agents tested) was obtained for 45 (77%) of 58 cultures. The overall error rate amounted to 1.9%, with the majority (1.3%) of errors being minor. Importantly, the results obtained with the direct method were available 12-24h earlier than those obtained with the current method.

Efficacy of Chromogenic Candida Agar for isolation and presumptive identification of pathogenic yeast species.

Chromogenic Candida Agar is a novel differential culture medium that is claimed to facilitate isolation and identification of Candida albicans, Candida tropicalis and Candida krusei. The performance of this medium was evaluated for presumptive identification of 521 yeast strains, representing 23 different species, for detection of specimens containing yeast mixtures, and for direct isolation of yeast from blood cultures. All yeasts grew well on the medium following a 48-h incubation period at 37 degrees C, and distinctive colonies were produced by C. albicans, C. tropicalis, C. krusei, Candida guilliermondii, Saccharomyces cerevisiae, Trichosporon mucoides and Geotrichum capitatum. The sensitivity and specificity of the medium exceeded 99.4% for each of these species. The medium provided some indication of the presence of Candida dubliniensis and Candida pulcherrima, and allowed the identification of polyfungal samples in 89.4% of the yeast mixtures. Finally, direct isolation on the medium from blood cultures that were positive for yeast according to Gram's stain (n = 42) showed that the expected colour and morphology of each species were not altered in the presence of blood.

Specific IgG4 response directed against the 45-kDa glycoprotein in trichinellosis: a re-evaluation of patients 15 years after infection.

The aim of this study was to evaluate the humoral immune response in late human trichinellosis with particular attention to the presence of IgG4 antibodies directed against the Trichinella-45-kDa glycoprotein (gp). This study re-evaluates subjects 15 years after they were involved in a trichinellosis outbreak that occurred in Central Italy following the consumption of raw boar meat infected with Trichinella britovi. The results show that ELISA tests using the E/S antigen identified five IgM- and eight IgG-positive patients and no IgA-positive patients. Tests using immunoblot (IB) with E/S antigens identified three IgM-, five IgA-, seven- IgG1- and three IgG4-positive sera. When the purified 45-kDa gp was used as an antigen, the IB revealed that six of the ten sera tested were positive for IgG4. Sera were also evaluated with a commercial kit, revealing that 11 of 12 patients had a highly sensitive reactivity against Trichinella proteins (64 and 44-43 kDa). In conclusion, humoral immune response against Trichinella is still present in these patients 15 years after the initial infection, including an IgG4 response directed to the 45-kDa gp.

Persistence of reactivity against the 45 k Da glycoprotein in late trichinellosis patients.

Over the years, the opinions of clinicians on the existence of the so-called chronic trichinellosis or late sequelae of infection have differed. However, the persistence of a humoral immune response against Trichinella in these late-stage patients has been confirmed using specific tests such as the competitive inhibition assay (CIA). We evaluated sera from late-stage trichinellosis patients (2--8 years from acute infection), for their reactivity against Trichinella spiralis antigens. The following tests were carried out: (i) indirect immunofluorescence assay (IFA), performed on muscle sections from mice, 30 days following synchronous infection by intramuscular injection with T. spiralis newborn larvae (NBL); (ii) enzyme immunoassay, employing a synthetic beta-tyvelose antigen conjugated to bovine serum albumin (BSA-Ag); and (iii) western blot (WB) with both an "in house" kit and a commercial kit. The results of IFA obtained by confocal laser microscopy showed that sera reacted against both surface and internal structures of L(1) larvae but at varying levels. Employing the synthetic antigen, EIA showed that 50% of sera tested were positive for the presence of specific antibodies against beta-tyvelose. By WB, all sera were reactive with the 45 k Da glycoprotein (45 gp). These data suggest that reactivity against the beta-tyvelosylated 45 gp persists even in very late stages of human trichinellosis.

Re-evaluation of patients involved in a trichinellosis outbreak caused by Trichinella britovi 15 years after infection.

This study re-evaluates 13 out of 48 subjects involved in a trichinellosis outbreak that occurred in Central Italy (Umbria Region) in 1988 resulting from the consumption of raw boar meat harboring Trichinella britovi. During the outbreak, 28 of 48 serologically positive subjects were asymptomatic, whereas 20 subjects presented one or more clinical signs including but not limited to fever, myalgia, periorbital oedema and conjunctivitis. Several patients were hospitalized with severe clinical signs requiring treatment with mebendazole and corticosteroids. Upon re-evaluation of 13 patients, none presented clinical signs; however, three still had increased CPK or LDH serum levels with some signs of electromyographic changes. In this study, enzyme immunoassays (EIA) were used to test the 13 positive sera for reactivity with T. britovi antigens using both excretory/secretory (E/S) antigens and a synthetic antigen composed of beta-tyvelose conjugated to bovine serum albumin. Western blots (WB) were also carried out using a commercial kit. Studies using EIA with E/S antigen identified five positive sera; however, using beta-tyvelose as antigen, only one positive sample was identified. Nearly all sera reacted positively with one or more Trichinella antigens when analyzed by WB, in particular to the 45 k Da beta-tyvelose containing glycoprotein. Results indicate that T. britovi, though less pathogenic than other Trichinella species, is clearly capable of inducing sustainable sequelae.

[The serodiagnosis of parasitic infections]. / La sierodiagnosi delle infezioni parassitarie.

Recently, the term of clinical immunoparasitology has been coined to indicate the application of immunological methods to the laboratory diagnosis of parasitic infections. In particular, serological diagnosis (indirect diagnosis) is useful especially in the cases of toxocarosis, trichinellosis, echinococcosis, cysticercosis, toxoplasmosis, amoebic abscess, some filariasis, visceral leishmaniasis, schistosomiasis. When possible, for infections caused by protozoa or helminths, the "gold standard" is represented by direct diagnosis performed by microscopic and/or macroscopic observation of the parasite. In any case, immunological results must be interpreted in consideration of the clinical picture of the patient and confirmed possibly by finding the parasite or its genome, even using molecular methods. Furthermore, since the presence of specific antibodies can reveal an acquired infection, but not necessarily a disease, it is particularly helpful, in addition to a qualitative evaluation, a quantitative one, by determining the serum antibody titre. After recovery, the antibody levels decrease, however, they may persist for long periods, for this reason they do not help in evaluating the treatment outcome. Interpretation of serological results may be difficult when the patients originate from areas where the suspected infection is endemic, in that case, a serum positivity could reflect an old exposition to the parasite, therefore it is not related to the present clinical status. Furthermore, serology may frequently result falsely negative in not immunocompetent subjects (organ transplanted, HIV positive individuals, premature babies, diabetics). Clinicians can interpret correctly the serological results only if the Parasitology laboratory inform them about the significant diagnostic values, the sensitivity and the specificity of the test in use. At present time, many diagnostic kits for immunoparasitology are commercially available, and industries are developing newer and newer ones (which are not always validated). In relation to this aspect, it should be helpful, for each of parasitic infection, to establish reference centers, not only to control the quality of commercial kits, but also as a reference point to those laboratories which use "in house" kits. To this regard, the recent establishment of a European Centre for Control of Infectious Diseases will help. The antigen characteristics (crude, E/S, recombinant, synthetic) for assays searching for antibodies (IHA, IFA, EIA, WB) of different classes, the controls to choose for these assays, the specimen requirements will be discussed. The recent findings on the serological diagnosis of intestinal protozoa infections, malaria, leishmaniasis, echinococcosis, cysticercosis, trichinellosis, toxocariasis, schistosomiasis, strongyloidiasis will be presented.

Characterization of antigens recognized by new monoclonal antibodies raised against culture filtrate proteins of Mycobacterium bovis bacillus Calmette-Guérin.

Effective protection against Mycobacterium tuberculosis may be achieved in experimental animals by immunization with proteins secreted by tuberculous bacilli in the extracellular milieu during growth. In this study, monoclonal antibodies were raised against Mycobacterium bovis bacillus Calmette-Guérin (BCG) culture filtrate proteins or live BCG, in an attempt to identify novel mycobacterial secretion antigens: the localization of the antigens recognized by the monoclonal antibodies within the mycobacterial cell was studied and interspecies reactivity was also investigated. The monoclonal antibodies obtained recognized proteins of molecular mass ranging from 5 to 82 kDa, with a prevailing frequency in the 30 kDa region. Three of the monoclonal antibodies recognized proteins present only in culture filtrates, one reacted with a cytoplasmic antigen, while the remaining antibodies recognized components which were mainly associated with the cell wall and the cytoplasmic membrane. The chemical nature and possible identity of the antigens was checked. Three monoclonal antibodies are likely to react with novel mycobacterial antigens of 5, 42 and 82 kDa, respectively.

Fibrin/fibrinogen and fibrinolytic activity of the palmar fascia in Dupuytren's contracture.

Considering the proved interaction of fibrin with fibroblasts and the seemingly decisive role of structural and functional changes ("modulation") of these cells in the evolution of Dupuytren's contracture, research has been carried out in order to investigate the fibrinolytic capacity and the possible presence of fibrin/fibrinogen in the palmar fascia of subjects operated upon for Dupuytren's Disease. Fibrin/fibrinogen were detected by a direct immunofluorescence technique and fibrinolytic activity was assessed by a fibrin plate method. A remarkable decrease of fibrinolytic activity and the presence of fibrin/fibrinogen were observed in small nodules in the early stage of disease, whereas large nodules showed a high amount of plasminogen activator enzymes. Small nodules seem to form and increase by progressive adhesion of fibroblasts to the polymerizing fibrin, while high fibrinolytic activity of large nodules probably results from "modulation" of many fibroblasts into contractile myofibroblasts and could therefore be considered as a biochemical sign of the evolutionary phase of Dupuytren's contracture.

Increased plasminogen activator enzymes of the tunica vaginalis in long-standing idiopathic hydrocele.

Previous research has shown increased fibrinolytic activity of the serous membranes as a reaction to stimuli that did not destroy their mesothelium. Large amounts of fluid remarkably stretch and chronically stimulate the tunica vaginalis in long-standing idiopathic hydrocele. For these reasons, a prospective controlled study was carried out in 30 male patients in order to investigate the possible alterations of the fibrinolytic capacity of this serosa by a method suitable for measuring plasminogen activator content of a single layer of cells. A possible role of plasminogen activator enzymes as a protective barrier against incidental absorption of fibrin/fibrinogen from the hydrocele fluid into vaginal tissues is considered.

Fibrinolytic activity of mesothelial lining of the displaced peritoneum.

Because of the suggestive analogy between a large hernial sac and the irreversibly displaced serosa, a prospective controlled study has been carried out in order to compare the fibrinolytic activity of mesothelial cells of the sac and of the parietal peritoneum in man. Results suggest the importance of undamaged mesothelial cells for the fibrinolytic activity of the peritoneal serosa. Careful defense of the mesothelial lining from direct trauma and damage has therefore to be regarded as necessary and perhaps sufficient requirement to preserve the fibrinolytic activity of attached peritoneal flaps dislocated to cover or separate the surgically deperitonealized surfaces.

[Anatomo-pathological study of the evolution of cryonecrosis in the rat pancreas]. / Studio anatomo-patologico dell'evoluzione di un focolaio di crionecrosi nel pancreas di ratto.

The progress of a cryonecrosis site occupying about one-third of the rat pancreas and induced with liquid nitrogen applied via a 10 mm luminal diameter probe applied for 2 min was followed. The lesion was not responsible for irreversible functional and anatomical lesions. Blood sugar remained within normal limits. Blood amylase peaked on the 1st day and was normal by the 14th. The treated area shrank considerably and was replaced by fibrous tissue within 30 days. The histological picture was similar to that encountered pathologically in man. Neither chain-type autolysis nor pseudocysts were observed.

[Anatomo-pathological study of the development of a focus of cryonecrosis in the rat spleen]. / Studio anatomo-patologico dell'evoluzione di un focolaio di crionecrosi nella milza di ratto.

A focus of cryonecrosis in the rat spleen, induced with liquid nitrogen (4 mm diameter probe, application time 2') does not cause haemorrhage either during or after treatment. The frozen area, which is of infarctual type with disappearance of spleen cells and preservation of the elastic structures of the follicular arteries, sees its surface reduced considerably and is replaced by connective tissue within 30 days.

Fibrinolytic activity of the human peritoneum.

A new method is presented to measure the content of plasminogen activator in single layers of mesothelial cells to investigate the fibrinolytic activity of the human peritoneum. The results demonstrate the synthesis of plasminogen activators in different areas of the peritoneal serosa. In the omental peritoneum, the fibrinolytic activity is significantly higher than in other parts of the peritoneum.