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Since the beginning of the pandemic, SARS-CoV-2 has caused problems for all of world’s population, not only in terms of deaths but also in terms of overloading healthcare facilities in all countries. Diagnosis is one of the key aspects of controlling the spread of SARS-CoV-2, and among the current molecular techniques, real-time PCR is considered as the gold standard. The availability of tests that allow for the rapid and accurate identification of SARS-CoV-2 is therefore of considerable importance. Moreover, if these tests allow for even minimal intervention by the operator, any risk of contamination is reduced. In this study, the performances of the new STANDARDTM M10 SARS-CoV-2 (SD Biosensor Inc., Suwon, Korea) rapid molecular test, which incorporates the above-mentioned features, were characterized. The clinical and analytical performances measured by testing different variants circulating in Italy of STANDARDTM M10 SARS-CoV-2 were compared to the test already on the market and recognized as the gold standard: Xpert Xpress SARS-CoV-2 (Cepheid, Sunnyvale, CA, USA). The results obtained between the two tests are largely comparable, suggesting that STANDARDTM M10 SARS-CoV-2 can be used with excellent results in the fight against the global spread of SARS-CoV-2.

Diagnostic tools that can rapidly identify and characterize microbes growing in blood cultures are important components of clinical microbiology practice because they help to provide timely information that can be used to optimize patient management. This publication describes the bioMérieux BIOFIRE Blood Culture Identification 2 (BCID2) Panel clinical study that was submitted to the U.S.

This work illustrates the development of a dry inhalation powder of cyclosporine-A for the prevention of rejection after lung transplantation and for the treatment of COVID-19. The influence of excipients on the spray-dried powder’s critical quality attributes was explored. The best-performing powder in terms of dissolution time and respirability was obtained starting from a concentration of ethanol of 45% (v/v) in the feedstock solution and 20% (w/w) of mannitol. This powder showed a faster dissolution profile (Weibull dissolution time of 59.5 min) than the poorly soluble raw material (169.0 min). The powder exhibited a fine particle fraction of 66.5% and an MMAD of 2.97 µm. The inhalable powder, when tested on A549 and THP-1, did not show cytotoxic effects up to a concentration of 10 µg/mL. Furthermore, the CsA inhalation powder showed efficiency in reducing IL-6 when tested on A549/THP-1 co-culture. A reduction in the replication of SARS-CoV-2 on Vero E6 cells was observed when the CsA powder was tested adopting the post-infection or simultaneous treatment. This formulation could represent a therapeutic strategy for the prevention of lung rejection, but is also a viable approach for the inhibition of SARS-CoV-2 replication and the COVID-19 pulmonary inflammatory process.

Methods of cell culture, viral propagation and PCR gene amplification.

Surgery of the gastrointestinal tract can result in deep changes among the gut commensals in terms of abundance, function and health consequences. Elective colorectal surgery can occur for neoplastic or inflammatory bowel disease; in these settings, microbiota imbalance is described as a preoperative condition, and it is linked to post-operative complications, as well. The study of bariatric patients led to several insights into the role of gut microbiota in obesity and after major surgical injuries. Preoperative dysbiosis and post-surgical microbiota reassessment are still poorly understood, and they could become a key part of preventing post-surgical complications. In the current review, we outline the most recent literature regarding agents and molecular pathways involved in pre- and post-operative dysbiosis in patients undergoing gastrointestinal surgery. Defining the standard method for microbiota assessment in these patients could set up the future approach and clinical practice.

Literature offers plenty of cases of immunocompromised patients, who develop chronic and severe SARS-CoV-2 infections. The aim of this study is to provide further insight into SARS-CoV-2 evolutionary dynamic taking into exam a subject suffering from follicular lymphoma, who developed a persistent infection for over 7 months. Eight nasopharyngeal swabs were obtained, and were analyses by qRT-PCR for diagnostic purposes. All of them were considered eligible (Ct < 30) for NGS sequencing. Sequence analysis showed that all sequences matched the B.1.617.2 AY.122 lineage, but they differed by few mutations identifying three genetically similar subpopulations, which evolved during the course of infection, demonstrating that prolonged replication is paralleled with intra-host virus evolution. These evidences support the hypothesis that SARS-CoV-2 adaptive capacities are able to shape a heterogeneous viral population in the context of immunocompromised patients. Spill-over of viral variants with enhanced transmissibility or immune escape capacities from these subjects is plausible.

This study aims to describe trends of mcr-positive Enterobacterales in humans based on laboratory surveillance with a defined catchment population. The data source is the Micro-RER surveillance system, established in Emilia-Romagna region (Italy), to monitor the trend of mcr resistance. Enterobacterales isolates from human clinical samples with minimum inhibitory concentration (MIC) ≥ 2mg/L for colistin were sent to the study reference laboratory for the detection of mcr genes. Isolates prospectively collected in the period 2018–2020 were considered for the assessment of population rates and trends; further analyses were carried out for the evaluation of clonality and horizontal mcr gene transfer. Previous isolates from local laboratory collection were also described. In the period 2018–2020, 1164 isolates were sent to the reference laboratory, and 51 (4.4%) were confirmed as mcr-positive: 50 mcr-1 (42 Escherichia coli, 6 Klebsiella pneumoniae, 2 Salmonella enterica) and 1 mcr-4 (Enterobacter cloacae). The number of mcr-positive isolates dropped from 24 in the first half of 2018 to 3 in the whole of 2020 (trend p value < 0.001). Genomic analyses showed the predominant role of the horizontal transfer of mcr genes through plasmids or dissemination of transposable elements compared to clonal dissemination of mcr-positive microorganisms. The study results demonstrate a substantial decrease in the circulation of mcr-1 plasmid genes in Emilia-Romagna Region.

Background: The pathogenetic steps leading to Covid-19 interstitial pneumonia remain to be clarified. Most postmortem studies to date reveal diffuse alveolar damage as the most relevant histologic pattern. Antemortem lung biopsy may however provide more precise data regarding the earlier stages of the disease, providing a basis for novel treatment approaches. Objectives: To ascertain the morphological and immunohistochemical features of lung samples obtained in patients with moderate Covid-19 pneumonia. Methods: Transbronchial lung cryobiopsy was carried out in 12 Covid-19 patients within 20 days of symptom onset. Results: Histopathologic changes included spots of patchy acute lung injury with alveolar type II cell hyperplasia, with no evidence of hyaline membranes. Strong nuclear expression of phosphorylated STAT3 was observed in >50% of AECII. Interalveolar capillaries showed enlarged lumen and were in part arranged in superposed rows. Pulmonary venules were characterized by luminal enlargement, thickened walls, and perivascular CD4+ T-cell infiltration. A strong nuclear expression of phosphorylated STAT3, associated with PD-L1 and IDO expression, was observed in endothelial cells of venules and interstitial capillaries. Alveolar spaces macrophages exhibited a peculiar phenotype (CD68, CD11c, CD14, CD205, CD206, CD123/IL3AR, and PD-L1). Conclusions: Morphologically distinct features were identified in early stages of Covid-19 pneumonia, with epithelial and endothelial cell abnormalities different from either classical interstitial lung diseases or diffuse alveolar damage. Alveolar type II cell hyperplasia was a prominent event in the majority of cases. Inflammatory cells expressed peculiar phenotypes. No evidence of hyaline membranes and endothelial changes characterized by IDO expression might in part explain the compliance and the characteristic pulmonary vasoplegia observed in less-advanced Covid-19 pneumonia.

Background and Objectives: Hemodialysis patients (HD) and kidney transplant recipients (KTRs) have been heavily impacted by COVID-19, showing increased risk of infection, worse clinical outcomes, and higher mortality rates than the general population. Although mass vaccination remains the most successful measure in counteracting the pandemic, less evidence is available on vaccine effectiveness in immunodepressed subjects previously infected and recovered from COVID-19. Materials and Methods: This study aimed at investigating the ability to develop an adequate antibody response after vaccination in a 2-dose series against SARS-CoV-2 in HD patients and KTR that was administered after laboratory and clinical recovery from COVID-19. Results: Comparing SARS-CoV-2 S1/S2 IgG levels measured before and after 2 doses of mRNA vaccine (BNT162b2 vaccine, Comirnaty, Pfizer–BioNTech or mRNA-1273 vaccine, Spikevax, Moderna), highly significant increases of antibody titers were observed. The antibody peak level was reached at 3 months following second dose administration, regardless of the underlying cause of immune depression and the time of pre-vaccine serology assessment after negativization. Conclusions: Our data indicate that HD patients and KTR exhibit a satisfying antibody response to a 2-dose series of mRNA vaccine, even in cases when infection-induced humoral immunity was poor or rapidly fading. Further studies are needed to evaluate the role of booster doses in conferring effective and durable protection in weak patient categories.

Gardnerella vaginalis (GV) is an anaerobic bacterial species involved in the pathogenesis of bacterial vaginosis (BV), a condition of vaginal dysbiosis associated with adverse pregnancy outcomes. GV strains are categorized into four clades, characterized by a different ability to produce virulence factors, such as sialidase. We investigated the distribution of GV clades and sialidase genes in the vaginal ecosystem of a cohort of pregnant women, assessing the correlations between GV clades and the whole vaginal microbiome. A total of 61 Caucasian pregnant women were enrolled. Their vaginal swabs, collected both at the first and third trimester of pregnancy, were used for (i) evaluation of the vaginal status by Nugent score, (ii) vaginal microbiome profiling by 16S rRNA sequencing, (iii) detection and quantification of GV clades and sialidase A gene by qPCR assays. DNA of at least one GV clade was detected in most vaginal swabs, with clade 4 being the most common one. GV clade 2, together with the presence of multiple clades (>2 simultaneously), were significantly associated with a BV condition. Significantly higher GV loads and sialidase gene levels were found in BV cases, compared to the healthy status. Clade 2 was related to the major shifts in the vaginal microbial composition, with a decrease in Lactobacillus and an increase in several BV-related taxa. As the number of GV clades detected simultaneously increased, a group of BV-associated bacteria tended to increase as well, while Bifidobacterium tended to decrease. A negative correlation between sialidase gene levels and Lactobacillus, and a positive correlation with Gardnerella, Atopobium, Prevotella, Megasphaera, and Sneathia were observed. Our results added knowledge about the interactions of GV clades with the inhabitants of the vaginal microbiome, possibly helping to predict the severity of BV and opening new perspectives for the prevention of pregnancy-related complications.

Background: Pleural empyema is associated with relevant morbidity and mortality, and it may be classified, according to evolution and ultrasound, into three stages: stage I (free-flowing effusion), stage II (viscous effusion with the tendency to loculate), and stage III (organizing phase). According to guidelines, antibiotic therapy and pleural drainage are recommended, with surgery being performed when patients fail and/or in case of organized empyema. Objectives: The aim of the study was to report the efficacy and safety of medical thoracoscopy in patients with pleural empyema stratified by chest ultrasound. Method: Observational retrospective cohort study analyzing patients with pleural empyema treated with medical thoracoscopy. Procedure success and mortality were evaluated at 30 days and 90 days after the procedure; complications were also reported. Results: 131 patients were included. Intrapleural fibrinolytic therapy was performed thereafter in the majority of cases. Medical thoracoscopy was considered successful without subsequent intervention in 99 patients (76%); 19 patients (15%) underwent a second procedure (drainage, thoracoscopy, video-assisted thoracic surgery, or thoracotomy); and 6 patients (5%) died of the evolution of empyema. Patients treated in stages I and II showed significantly better post-procedure results compared with patients treated in stage III (100%, 83.3%, and 58.1%, respectively). Thoracoscopy complications were observed in 18 patients and were reversible in all cases. Conclusions: Patients with pleural empyema treated in earlier stages (free-flowing or multiloculated effusion) with medical thoracoscopy show significantly better results than patients treated in later stages (organized empyema). This approach is safe, minimally invasive, and efficient in these patients with disease having relevant mortality; however, patient selection remains essential.

The level of One Health (OH), or “One Health-ness,” of health interventions has been defined as the capacity to operate according to six dimensions concerning OH operations and OH infrastructures, respectively (thinking, planning, and working; and information sharing, reciprocal learning, and systemic organization). Although health initiatives and research increasingly claim their orientation toward OH, such a capacity is rarely assessed. The objective of this study is to evaluate the One Health-ness of the academic team of the University of Bologna (UNIBO Team) working in the “ELEPHANT” project (Empowering universities' Learning and rEsearch caPacities in the one Health Approach for the maNagement of animals at the wildlife, livestock and human interface in SouTh Africa). This project involves universities, six from South Africa and two from Europe, and aims at embedding OH in research and learning to enable the control of diseases at the human, animal, and environmental interface, and to emphasize the interests of local African communities with wildlife conservation. The methodology adopts the NEOH method, developed in 2018 by the EU-COST Action, “Network for the Evaluation of One Health.” The approach is based on questionnaires delivered to participants, which focus on the six OH dimensions, and then translate answers into quantitative metrics through the OH Index (OHI) and the OH Ratio (OHR). The following two evaluation levels are foreseen: the whole project and the single partner institutions. The evaluations are carried on in parallel, with preliminary, mid-term, and final assessments, to monitor the efficacy of the project actions. The preliminary evaluation of the UNIBO Team resulted in the OHI of 0.23 and the OHR of 1.69 which indicate a low degree of OH-ness and an imbalance between OH operation and OH infrastructure. The UNIBO case study will be the baseline for the evaluation of the other partner institutions involved in the ELEPHANT project. This type of evaluation can support the implementation of OH practices inside a project and underpin the strategies that allow to achieving more effective results. Any improvement in the OH-ness of each single academic team can be also considered as a result of the ELEPHANT project, thus showing its multiplier effect in the context.

Aging results in a progressive decline in skeletal muscle mass, strength and function, a condition known as sarcopenia. This pathological condition is due to multifactorial processes including physical inactivity, inflammation, oxidative stress, hormonal changes, and nutritional intake. Physical therapy remains the standard approach to treat sarcopenia, although some interventions based on dietary supplementation are in clinical development. In this context, thanks to its known anti-inflammatory and antioxidative properties, there is great interest in using extra virgin olive oil (EVOO) supplementation to promote muscle mass and health in sarcopenic patients. To date, the molecular mechanisms responsible for the pathological changes associated with sarcopenia remain undefined; however, a complete understanding of the signaling pathways that regulate skeletal muscle protein synthesis and their behavior during sarcopenia appears vital for defining how EVOO might attenuate muscle wasting during aging. This review highlights the main molecular players that control skeletal muscle mass, with particular regard to sarcopenia, and discusses, based on the more recent findings, the potential of EVOO in delaying/preventing loss of muscle mass and function, with the aim of stimulating further research to assess dietary supplementation with EVOO as an approach to prevent or delay sarcopenia in aging individuals.

Bioaerosols (aerosolized particles with biological origin) are strongly suspected to play a significant role in the transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), especially in closed indoor environments. Thus, control technologies capable of effectively inactivating bioaerosols are urgently needed. In this regard, cold atmospheric pressure plasma (CAP) can represent a suitable option, thanks to its ability to produce reactive species, which can exert antimicrobial action. In this study, results; on the total inactivation of SARS-CoV-2 contained in bioaerosols treated using CAP generated in air are reported, demonstrating the possible use of CAP systems for the control of SARS-CoV-2 diffusion through bioaerosols.

Simple Summary Pancreatic cancer is still burdened with a severe prognosis, despite advances in the diagnosis and surgical management of this disease. The gut microbiome is gaining increasing interest in the development and management in this setting. The intent of our review is to provide a comprehensive review for researchers and clinicians in the field to fully understand the role of the gut microbiome in the history of pancreatic cancer. We analyzed current literature from pre-cancerous conditions to cancer characteristics and how this may alter the therapeutic approach. Evidence and concerns can guide future research in this area. Early microbiome insights came from gut microbes and their role among intestinal and extraintestinal disease. The latest evidence suggests that the microbiota is a true organ, capable of several interactions throughout the digestive system, attracting specific interest in the biliopancreatic district. Despite advances in diagnostics over the last few decades and improvements in the management of this disease, pancreatic cancer is still a common cause of cancer death. Microbiota can influence the development of precancerous disease predisposing to pancreatic cancer (PC). At the same time, neoplastic tissue shows specific characteristics in terms of diversity and phenotype, determining the short- and long-term prognosis. Considering the above information, a role for microbiota has also been hypothesized in the different phases of the PC approach, providing future revolutionary therapeutic insights. Microbiota-modulating therapies could open new issues in the therapeutic landscape. The aim of this narrative review is to assess the most updated evidence on microbiome in all the steps regarding pancreatic adenocarcinoma, from early development to response to antineoplastic therapy and long-term prognosis.

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Recent evidence regarding microbiota is modifying the cornerstones on pathogenesis and the approaches to several gastrointestinal diseases, including biliary diseases. The burden of biliary diseases, indeed, is progressively increasing, considering that gallstone disease affects up to 20% of the European population. At the same time, neoplasms of the biliary system have an increasing incidence and poor prognosis. Framing the specific state of biliary eubiosis or dysbiosis is made difficult by the use of heterogeneous techniques and the sometimes unwarranted invasive sampling in healthy subjects. The influence of the microbial balance on the health status of the biliary tract could also account for some of the complications surrounding the post-liver-transplant phase. The aim of this extensive narrative review is to summarize the current evidence on this topic, to highlight gaps in the available evidence in order to guide further clinical research in these settings, and, eventually, to provide new tools to treat biliary lithiasis, biliopancreatic cancers, and even cholestatic disease.

The inhabitants of the vaginal ecosystem can harbor genetic determinants conferring antimicrobial resistance. However, detailed data about the distribution of resistance genes in the vaginal microbiome of pregnant women are still lacking. Therefore, we assessed the presence of macrolide (i.e., erm genes) and tetracycline (i.e., tet genes) resistance markers in the vaginal environment of Caucasian women at different gestational ages. Furthermore, the detection of resistance genes was related to the composition of the vaginal microbiota. A total of 228 vaginal samples, collected at different trimesters of pregnancy or during the puerperium, were tested for the presence of ermB, ermF, tet(W), and tet(M) by in-house end-point PCR assays. The composition of the vaginal microbiota was assessed through a microscopic evaluation (i.e., Nugent score) and by means of sequencing V3–V4 hypervariable regions of the bacterial 16 rRNA gene. Overall, the most detected resistance gene was tet(M) (76.7%), followed by ermB (55.2%). In 17% of women, mainly with a ‘normal’ vaginal microbiota, no resistance genes were found. Except for tet(W), a significant correlation between the positivity of resistance genes and a dysbiotic vaginal status (i.e., bacterial vaginosis (BV)) was noticed. Indeed, samples positive for at least one resistance determinant were characterized by a decrease in Lactobacillus spp. and an increase of BV-related genera (Prevotella, Gardnerella, Atopobium, Sneathia). A high predominance of vaginal Lactobacillus spp. (>85%) was associated with a lower risk of tet(W) gene detection, whereas the presence of Megasphaera (>1%) increased the risk of positivity for all analyzed genes. Different types of vaginal microbiota are associated with peculiar resistance profiles, being a lactobacilli-dominated ecosystem poor in or free of resistance genes. These data could open new perspectives for promoting maternal and neonatal health.

Nephropathic subjects with impaired immune responses show dramatically high infection rates of coronavirus disease 2019 (COVID-19). This work evaluated the ability to acquire and maintain protective antibodies over time in 26 hemodialysis patients and 21 kidney transplant recipients. The subjects were followed-up through quantitative determination of circulating SARS-CoV-2 S1/S2 IgG and neutralizing antibodies in the 6-month period after clinical and laboratory recovery. A group of 143 healthcare workers with no underlying chronic pathologies or renal diseases recovered from COVID was also evaluated. In both dialysis and transplanted patients, antibody titers reached a zenith around the 3rd month, and then a decline occurred on average between the 270th and 300th day. Immunocompromised patients who lost antibodies around the 6th month were more common than non-renal subjects, although the difference was not significant (38.5% vs. 26.6%). Considering the decay of antibody levels below the positivity threshold (15 AU/mL) as “failure”, a progressive loss of immunisation was found in the overall population starting 6 months after recovery. A longer overall antibody persistence was observed in severe forms of COVID-19 (p = 0.0183), but within each group, given the small number of patients, the difference was not significant (dialysis: p = 0.0702, transplant: p = 0.1899). These data suggest that immunocompromised renal patients recovered from COVID-19 have weakened and heterogeneous humoral responses that tend to decay over time. Despite interindividual variability, an association emerged between antibody persistence and clinical severity, similar to the subjects with preserved immune function.

Background COVID-19 severity is uneven between genders. We hypothesized a role of hormonal therapies in the severity of COVID-19 in breast cancer (BC) patients via the modulation of SARS-CoV-2 susceptibility genes. Patients and Methods We mined the Emilia Romagna region (Italy) registries to compare the rates of hospitalization and mortality for COVID-19 in 2020 amongst 24628 BC patients. Next, we analyzed the modulation of ACE2, TMPRSS2 and NRP1 gene expression and the susceptibility to SARS-CoV-2 infection by tamoxifen, fulvestrant and 17β-estradiol on human ER+ MCF-7 cells in vitro.Results The hospitalization rate observed for 4784 tamoxifen treated BC patients was the lowest (OR, 0.41; 95% CI, 0.18-0.94; p=0.04) among hormonal therapies and no fatalities occurred. A standard mortality rate reduction has been observed also for patients treated with aromatase inhibitors (SMR: 0.73; 95% CI, 0.45-0.90). In vitro experiments showed that fulvestrant, but not tamoxifen, increases ACE2, TMPRSS2 and NRP1 gene expression and susceptibility to SARS-CoV-2 infection and that 17β-estradiol reduces significantly TMPRSS2 and NRP1 expression.Conclusions Tamoxifen treated BC patients showed a reduced rate of hospitalization and strikingly no fatalities for COVID-19. In vitro experiments confirmed a protective role of tamoxifen while an increased susceptibility to SARS-CoV-2 infection of ER+ cells treated with fulvestrant was observed.

Community-acquired pneumonia (CAP) is one of the major causes of morbidity, mortality and hospitalization, and S. pneumoniae is the most frequently isolated etiologic agent. The pneumococcal urinary antigen test (PUAT) is among the recommended methods to identify the causative agent in CAP patients. A novel PUAT (IMMUNOCATCHTMStreptococcus pneumoniae) was compared with the Uni-GoldTMS. pneumoniae assay routinely used in our laboratory and with the widely used BinaxNOW® S. pneumoniae antigen card. A total of 218 (183 freshly harvested and 35 frozen) urine samples (US) submitted for the detection of pneumococcal urinary antigen (PUAT) between December 2016 and November 2018 were evaluated. A number of 160 negative and 41 positive concordant results were scored for all the three assays. A total of 17 US gave discrepant results. The sensitivity and specificity of Immunocatch compared with Uni-Gold were 73.2% and 98.8%, respectively, and compared with BinaxNOW were 97.6% and 98.8%, respectively. The overall percent agreement (OPA) and the Cohen’s kappa coefficient between the Immunocatch and the Uni-Gold resulted 92.2% and 0.78%, respectively, and compared with BinaxNOW were 98.6% and 0.95%, respectively. These performances suggest that the novel Immunocatch S. pneumoniae test is a useful tool for qualitative detection of S. pneumoniae capsular antigen in US.

The Unyvero molecular assay was tested for the clinical resolution of discordant results, evaluating its role in prosthetic joint infection diagnosis. Objectives: The Unyvero molecular assay was tested for the clinical resolution of discordant results, evaluating its role in prosthetic joint infection diagnosis. Methods: Multiplex PCR was performed on 45 samples from prosthesis treatment (either sonication or dithiothreitol). Analytical performance was compared to that of biofilm culture using Musculoskeletal Infection Society criteria as gold standard. Results: Unyvero and biofilm culture showed similar agreement rates compared to the gold standard (34/43 and 32/43, respectively). Both methods showed six additional identifications compatible with true infection; five positive results from biofilm culture were deemed contaminations. Conclusions: The Unyvero system showed good performances and a significantly shorter turnaround time compared to cultural methods, presenting an added value to PJI diagnosis even when performed following a composite approach.

The incidence of total joint arthroplasty is increasing over time since the last decade and expected to be more than 4 million by 2030. As a consequence, the detection of infections associated with surgical interventions is increasing and prosthetic joint infections are representing both a clinically and economically challenging problem. Many pathogens, from bacteria to fungi, elicit the immune system response and produce a polymeric matrix, the biofilm, that serves as their protection. In the last years, the implementation of diagnostic methodologies reduced the error rate and the turn-around time: polymerase chain reaction, targeted or broad-spectrum, and next-generation sequencing have been introduced and they represent a robust approach nowadays that frees laboratories from the unique approach based on culture-based techniques.

The immune system is a dynamic feature of each individual and a footprint of our unique internal and external exposures. Indeed, the type and level of exposure to physical and biological agents shape the development and behavior of this complex and diffuse system. Many pathological conditions depend on how our immune system responds or does not respond to a pathogen or a disease or on how the regulation of immunity is altered by the disease itself. T-cells are important players in adaptive immunity and, together with B-cells, define specificity and monitor the internal and external signals that our organism perceives through its specific receptors, TCRs and BCRs, respectively. Today, high-throughput sequencing (HTS) applied to the TCR repertoire has opened a window of opportunity to disclose T-cell repertoire development and behavior down to the clonal level. Although TCR repertoire sequencing is easily accessible today, it is important to deeply understand the available technologies for choosing the best fit for the specific experimental needs and questions. Here, we provide an updated overview of TCR repertoire sequencing strategies, providers and applications to infectious diseases and cancer to guide researchers’ choice through the multitude of available options. The possibility of extending the TCR repertoire to HLA characterization will be of pivotal importance in the near future to understand how specific HLA genes shape T-cell responses in different pathological contexts and will add a level of comprehension that was unthinkable just a few years ago.

During pregnancy, the vaginal ecosystem undergoes marked changes, including a significant enrichment with Lactobacillus spp. and profound alterations in metabolic profiles. A deep comprehension of the vaginal environment may shed light on the physiology of pregnancy and may provide novel biomarkers to identify subjects at risk of complications (e.g., miscarriage, preterm birth). In this study, we characterized the vaginal ecosystem in Caucasian women with a normal pregnancy (n = 64) at three different gestational ages (i.e., first, second and third trimester) and in subjects (n = 10) suffering a spontaneous first trimester miscarriage. We assessed the vaginal bacterial composition (Nugent score), the vaginal metabolic profiles (1H-NMR spectroscopy) and the vaginal levels of two cytokines (IL-6 and IL-8). Throughout pregnancy, the vaginal microbiota became less diverse, being mainly dominated by lactobacilli. This shift was clearly associated with marked changes in the vaginal metabolome: over the weeks, a progressive reduction in the levels of dysbiosis-associated metabolites (e.g., biogenic amines, alcohols, propionate, acetate) was observed. At the same time, several metabolites, typically found in healthy vaginal conditions, reached the highest concentrations at the end of pregnancy (e.g., lactate, glycine, phenylalanine, leucine, isoleucine). Lower levels of glucose were an additional fingerprint of a normal vaginal environment. The vaginal levels of IL-6 and IL-8 were significantly associated with the number of vaginal leukocytes, as well as with the presence of vaginal symptoms, but not with a condition of dysbiosis. Moreover, IL-8 concentration seemed to be a good predictor of the presence of vaginal Candida spp. Cytokine concentrations were negatively correlated to lactate, serine, and glycine concentrations, whereas the levels of 4-hydroxyphenyllactate, glucose, O-acetylcholine, and choline were positively correlated with Candida vaginal loads. Finally, we found that most cases of spontaneous abortion were associated with an abnormal vaginal microbiome, with higher levels of selected metabolites in the vaginal environment (e.g., inosine, fumarate, xanthine, benzoate, ascorbate). No association with higher pro-inflammatory cytokines was found. In conclusion, our analysis provides new insights into the pathophysiology of pregnancy and highlights potential biomarkers to enable the diagnosis of early pregnancy loss.

Vaginal microbes and their metabolic products have crucial functions, affecting local immunity development and maternal-fetal health. The composition of the vaginal microbiome can vary in response to various factors, including body mass index (BMI), and diet. In this study we get new insights into the vaginal ecosystem of Caucasian women (n = 24) at the first trimester of pregnancy, assessing whether pre-pregnancy diet can affect the structure of the vaginal environment in terms of bacterial composition and vaginal metabolite concentration. We characterized 1) the vaginal bacterial composition (Nugent score), 2) the vaginal metabolic profiles (1H-NMR spectroscopy), and 3) the dietary nutrient intake by means of a validated food frequency questionnaire. Pre-pregnancy BMI was negatively related to vaginal health status, indicating that women who begin pregnancy overweight/obese have a greater occurrence of vaginal dysbiosis during pregnancy. A lactobacilli-dominated vaginal microbiota was negatively associated with higher pre-pregnancy intake of animal-sourced protein. Conversely, a higher pre-pregnancy consumption of total carbohydrates and sugars seemed to be a protective factor for vaginal health. The vaginal environment of BV-women was characterized by higher levels of biogenic amines and organic acids, whereas higher levels of phenylpropionate and diverse amino acids were fingerprints of a healthy vaginal status. A significant association between a higher pre-pregnancy BMI and several dysbiosis-related vaginal metabolites was also found. Our study shed light on the role of pre-pregnancy BMI and diet on the vaginal environment during pregnancy, underlining the importance of limiting protein intake from animal foods to maintain a healthy lactobacilli-dominated microbiota.

IntroductionMutations in the receptor binding domain (RBD) region of SARS-CoV-2 have been shown to impact the infectivity, pathogenicity and transmissibility of new variants of concern (VOC). Even more worrisome, those mutations have the potential of causing immune escape, undermining the population immunity induced by ongoing mass vaccination programs.Gap statementThe massive parallel sequencing techniques have taken a lead role in the detection strategies of the new variants. Nevertheless, they are still cumbersome and labour-demanding. There is an urgent need for novel strategies and techniques aimed at the surveillance of the active emergence and spread of the VOC.AimThe aim of this study was to provide a quick, cheap and straightforward Denaturing High-Performance Liquid Chromatography (DHPLC) method for the prompt identification of the SARS-CoV-2 VOC.MethodologyTwo PCRs were designed to target the RBD region, spanning residues N417 through N501 of the Spike protein. Furthermore, a DHPLC screening analysis was set up. The screening consisted of mixing the unknown sample with a standard sample of a known variant, denaturing at high temperature, renaturing at room temperature followed by a 2-minute run using the WAVE DHPLC system to detect the heteroduplexes which invariably originate whenever the unknown sample has a nucleotide difference with respect to the standard used.ResultsThe workflow was able to readily detect new variants including the P.1, the B.1.585 and the B.1. 617.2 lineages at a very affordable cost. The DHPLC analysis was robust being able to identify variants even in case of samples with very unbalanced target concentration including those samples at the limit of detection.ConclusionsThis approach has the potential of greatly expediting surveillance of the SARS-CoV-2 variants.

Purpose To investigate the prevalence, incidence and characteristics of bacterial infections and their impact on outcome in critically ill patients infected with COVID-19. Methods We conducted a prospective observational study in eight Italian ICUs from February to May 2020; data were collected through an interactive electronic database. Kaplan–Meier analysis (limit product method) was used to identify the occurrence of infections and risk of acquisition. Results During the study period 248 patients were recruited in the eight participating ICUs. Ninety (36.3%) patients developed at least one episode of secondary infection. An ICU length of stay between 7 and 14 days was characterized by a higher occurrence of infectious complications, with ventilator-associated pneumonia being the most frequent. At least one course of antibiotic therapy was given to 161 (64.9%) patients. Overall ICU and hospital mortality were 33.9% and 42.9%, respectively. Patients developing bacteremia had a higher risk of ICU mortality [45.9% vs. 31.6%, odds ratio 1.8 (95% CI 0.9–3.7), p = 0.069] and hospital mortality [56.8% vs. 40.3%, odds ratio 1.9 (95% CI 1.1–3.9), p = 0.04]. Conclusion In critically ill patients infected with COVID-19 the incidence of bacterial infections is high and associated with worse outcomes. Regular microbiological surveillance and strict infection control measures are mandated. Supplementary Information The online version contains supplementary material available at 10.1007/s15010-021-01661-2.

In-vitro viral studies are still fundamental for biomedical research since studying the virus kinetics on cells is crucial for the determination of the biological properties of viruses and for screening the inhibitors of infections. Moreover, testing potential viral contaminants is often mandatory for safety evaluation. Nowadays, viral cytopathic effects are mainly evaluated through end-point assays requiring dye-staining combined with optical evaluation. Recently, optical-based automatized equipment has been marketed, aimed at the real-time screening of cell-layer status and obtaining further insights, which are unavailable with end-point assays. However, these technologies present two huge limitations, namely, high costs and the possibility to study only cytopathic viruses, whose effects lead to plaque formation and layer disruption. Here, we employed poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (Pedot:Pss) organic electrochemical transistors (OECTs) for the real-time, electrical monitoring of the infection of cytolytic viruses, i.e., encephalomyocarditis virus (EMCV), and non-cytolytic viruses, i.e., bovine coronavirus (B-CoV), on cells. OECT data on EMCV were validated using a commercially-available optical-based technology, which, however, failed in the B-CoV titration analysis, as expected. The OECTs proved to be reliable, fast, and versatile devices for viral infection monitoring, which could be scaled up at low cost, reducing the operator workload and speeding up in-vitro assays in the biomedical research field.

In most children, coronavirus disease 2019 (COVID-19) is a mild or moderate disease. Moreover, in a relevant number of cases, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains totally asymptomatic. All these findings seem to suggest that otherwise healthy children with suspected COVID-19 might be managed in the community in most cases, thus avoiding hospital admission and closely related medical, social and economic problems, including overwhelming hospitals. Unfortunately, home management of children with suspected COVID-19 rarely occurs, and many children with suspected or laboratory-confirmed SARS-CoV-2 infection are frequently hospitalized irrespective of the severity of disease. To evaluate the role of community health houses (CHHs) in the management of children with COVID-19, 1,009 children with suspected SARS-CoV-2 infection were studied in Emilia-Romagna Region, Italy. Among them, 194 (19.2%) resulted positive for SARS-CoV-2. The majority (583, 58%) were tested at home by CHHs, while 426 (42%) were brought to the hospital for testing. The patients who were managed in the hospital had a significantly lower median age than those who were managed at home (2 vs. 12 years, p < 0.001). Exposure to SARS-CoV-2 cases within the family was significantly more frequent among those who were managed at home (82 vs. 46%, p < 0.05). The clinical findings were similar between the children who were managed at home and those who were managed in the hospital. Only one of the children managed at home (0.7%) required hospitalization; in comparison, 26 (48%) of those whose swab samples were taken at the hospital were hospitalized. Our research shows for the first time the importance of CHHs in the management of COVID-19 in children; because of the high frequency of mild to moderate cases, management by CHHs can reduce the care load in hospitals, providing enormous advantages on the familial, medical, social, and economic levels. These findings could be useful for suggesting a territorial rather than hospital-based strategy in pediatrics in the case of a new wave of the epidemic.

The number of total knee and/or hip replacements are expected to exceed 5 million a year by 2030; the incidence of biofilm-associated complications can vary from 1% in primary implants to 5.6% in case of revision. The purpose of this study was to test the ability of sHA-DA, a partially sulphated hyaluronic acid (sHA) functionalized with a dopamine (DA) moiety, to prevent acute bacterial growth in an in vivo model of an intra-operatively highly contaminated implant. Previously, in vitro studies showed that the DA moiety guarantees good performance as binding agent for titanium surface adhesion, while the negatively charged sHA has both a high efficiency in electrostatic binding of positively charged antibiotics, and bone regenerative effects. The in vitro testing also highlighted the effectiveness of the sHA-DA system in inhibiting bacterial spreading through a sustained release of the antibiotic payload from the implant coating. In this study the chemical stability of the sHA-DA to β-ray sterilization was demonstrated, based on evaluation by NMR, SEC-TDA Omnisec and HPLC-MS analysis, thus supporting the approach of terminal sterilization of the coated implant with no loss of efficacy. Furthermore, an in vivo study in rabbits was performed according to UNI EN ISO 10993-6 to assess the histocompatibility of titanium nails pre-coated with sHA-DA. The implants, placed in the femoral medullary cavity and harvested after 12 weeks, proved to be histocompatible and to allow bone growth in adhesion to the metal surface. Finally, an in vivo model of bacterial contamination was set up by injecting 1 mL of bacterial suspension containing 104 or 106 CFU of methicillin-resistant Staphylococcus aureus (MRSA) into the femoral medullary cavity of 30 rabbits. Titanium nails either uncoated or pre-coated with sHA-DA and loaded directly by the surgeon with 5% vancomycin were implanted in the surgical site. After 1 week, only the animals treated with pre-coated nails did not show the presence of systemic or local bacterial infection, as confirmed by microbiology and histology (Smeltzer score). Further insights into the animal model setup are crucial, however the results obtained suggest that the system can be effective in preventing the onset of the bacterial infective process.

We describe the establishment and current content of the ImmuneCODE™ database, which includes hundreds of millions of T-cell Receptor (TCR) sequences from over 1,400 subjects exposed to or infected with the SARS-CoV-2 virus, as well as over 135,000 high-confidence SARS-CoV-2-specific TCRs. This database is made freely available, and the data contained in it can be downloaded and analyzed online or offline to assist with the global efforts to understand the immune response to the SARS-CoV-2 virus and develop new interventions.

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The laboratory diagnosis of STDs caused by bacteria and protozoa has been in place since the identification of the specific pathogens. Initially, the culture based techniques and the microscopic examination of genital secretions were considered the reference methods and in selected cases these methods are still relevant to achieve the laboratory diagnosis. In the last decades the major improvements in the diagnostic workflows were made based on the use of recombinant antigens for the serology of syphilis and the implementation of nucleic acids amplification techniques for most of these infections. The laboratory diagnosis of syphilis, Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and genital mycoplasma is discussed, with a focus on the most innovative approaches.

Shewanella algae are Gram-negative, nonfermentative, motile bacilli, classified in the genus Shewanella in 1985. These environmental bacteria are occasionally identified in human infections, with a relatively strong association with exposure to seawater during warm seasons. This report describes a case series of 17 patients with infection correlated to S. algae in the coastal area of Romagna, Italy, from 2013 to 2016. The types of infection included otitis, pneumonia, sepsis and soft tissue (wound). Exposure to the marine environment during hot months was confirmed in 12 of 17 patients. An apparent correlation between increased severity of infection and patient age was also observed. Keywords: Adriatic Sea, otitis, seawater, sepsis, Shewanella algae

Chronic HCV infection is one of the leading causes of liver-related death and in many countries it is a primary reason for having a liver transplant. HCV genotype identification has long been used in the clinical practice, since different genotypes have different response rates and required different doses and durations of IFN/RBV treatment; moreover both the frequency and the pattern of resistance to different Direct-Acting Antivirals (DAAs) classes are subtype specific. Hence the necessity to make an accurate HCV subtyping becomes a fundamental tool to optimize current and future clinical management of HCV infected subjects. In the present study the performance of a next generation sequencing (NGS: based on the Ion Torrent Platform-Vela Sentosa SQ 301 sequencer) HCV genotyping assay has been evaluated. The current method targets a region of the NS5 B gene and it is the unique NGS based market CE-IVD assay. As a comparative method a commercial method based on the detection via reverse hybridization of 5′UTR and core regions (Versant HCV Genotype 2.0 Assay, LiPA, Siemens) was selected. A total 207 plasma samples from HCV infected individuals were used. No selection was made for these samples that were submitted for routine HCV genotyping. The results show Vela NGS assay assigns major number of HCV subtypes with respect LiPA. Concerning genotype 1 and 3, the discrepancy of assigned subtypes for LiPA with respect to Vela NGS assay is not relevant (1.8% and 2%, respectively); in contrast, the difference of assigned subtypes for genotypes 2 and 4 is very high (96.6% and 100%, respectively). The resistance mutations data, except for 1a and 1b subtypes, remain scarce; the future relevant challenge will be to identify subtypes-specific drug resistance mutations, which are essential to create highly personalized therapeutic pathways.

Background There are no Italian data regarding the strategies for preventing neonatal group B streptococcal (GBS) infection. We conducted a national survey in order to explore obstetrical, neonatal and microbiological practices for the GBS prevention. Methods Three distinct questionnaires were sent to obstetricians, neonatologists and microbiologists. Questionnaires included data on prenatal GBS screening, maternal risk factors, intrapartum antibiotic prophylaxis, microbiological information concerning specimen processing and GBS antimicrobial susceptibility. Results All respondent obstetrical units used the culture-based screening approach to identify women who should receive intrapartum antibiotic prophylaxis, and more than half of the microbiological laboratories (58%) reported using specimen processing consistent with CDC guidelines. Most neonatal units (89 out of 107, 82%) reported using protocols for preventing GBS early-onset sepsis consistent with CDC guidelines. Conclusions The screening-based strategy is largely prevalent in Italy, and most protocols for preventing GBS early-onset sepsis are consistent with CDC guidelines. However, we found discrepancies in practices among centers that may reflect the lack of Italian guidelines issued by public health organizations. Electronic supplementary material The online version of this article (10.1186/s13052-017-0409-1) contains supplementary material, which is available to authorized users.

Chikungunya fever epidemics display secular, cyclical, and seasonal trends. These epidemics are characterized by explosive outbreaks inter- spersed by periods of disappearance ranging from several years to a few decades. Several mechanisms play a role: the human and the mosquito vector susceptibility to the virus; conditions facilitating mosquito breed- ing (resulting in a high vector density), ability of the vector to efficiently transmit the virus. There are historical accounts of epidemics of fever, arthralgias/arthritis, and rash, resembling what is now called “Chikungunya fever” dating back to 1824 from India and elsewhere (Kaur et al., 2008). Chikungunya virus (CHIKV) was firstly detected in Central/East Africa, where it is maintained in a sylvatic transmission cycle between nonhuman primates, small mammals (e.g., bats and monkeys) and Aedes mosquitoes . Urban chikungunya fever outbreaks are initiated by spillover infection of humans from enzootic African transmission cycles. The first identified outbreak of Chikungunya was reported from July 1952 to March 1953 in Tanzania. Since its discovery, numerous Chikungunya re-emergences have been documented. Currently, the Chikungunya virus has been identified in over 60 countries. The risk of importation of CHIKV into new areas is ever present because of the high attack rates associated with the recurring epidemics, the high levels of vi- remia in infected humans, and the worldwide distribution of the vectors responsible for transmitting CHIKV. E1-A226V and E2-L210Q mutations have been found to cause a dramatic increase in the infectivity of CHIKV, and the transmission of CHIKV has spread to Europe and the Americas because of the widespread distribution of the vectors Aedes aegypti and Ae. albopictus

Objectives The early detection of bacteraemia and fungemia is of paramount importance to guide antimicrobial therapy in septic patients. In this study the ‘time to detection' (TTD) value for the new blood culture system BacT/ALERT VIRTUO (VIRTUO) was evaluated in 1462 positive clinical bottles and compared with the TTD for 1601 positive clinical bottles incubated in the BacT/ALERT 3D system (BTA-3D). Methods The most representative microorganisms isolated from bottles incubated in both blood culture systems were divided into eight categories (in order of frequency): coagulase-negative staphylococci (CoNS), Escherichia coli, Enterobacteriaceae (other than E. coli ), Staphylococcus aureus, Enterococcus spp , viridans group streptococci, Pseudomonas aeruginosa , and Candida spp . Results The comparison of TTD values for the two blood culture systems strongly indicated that growth of the first five groups listed above was detected earlier with VIRTUO than with BTA-3D ( p Conclusions The new VIRTUO blood culture system can reduce the TTD for more than 75% of isolated microorganisms.

Objectives: To start a surveillance program to investigate the possible diffusion of mobilized colistin resistance genes in Enterobacteriaceae strains isolated in the Unit of Microbiology of the Great Romagna Hub Laboratory. Methods: All the colistin resistant Enterobacteriaceae, isolated from August 1st 2016 to July 31st 2017, were prospectively evaluated for mcr-1 and mcr-2. Backdated survey of mcr-3, mcr-4 and mcr-5 was performed on the same group of isolates. Species identification was achieved by Vitek MS and the antibiotic susceptibility testing was performed both with Vitek-2 and Sensititre systems. Colistin resistant isolates were screened by PCR for the presence of the plasmid-mediated colistin resistance genes and amplicons were verified by sequencing. All mcr-1 positive isolates were subjected to MLST analysis. Results: Over the total of 19053 isolates belonging to Enterobacteriaceae, 90 were colistin resistant. The presence of mcr-1 was detected in 26 Escherichia coli. The overall prevalence of mcr-1 was 0.14%. The mcr-1 positive E. coli strains were assigned to 13 distinct sequence types (STs) according to MLST. Conclusions: The prospective epidemiological survey carried out in our study gave a glimpse of the plasmid-mediated colistin resistance dissemination in Romagna. Since the prevalence rate of carbapenem resistant Enterobacteriaceae (CRE) in some hospital wards in our area is alarming, we underline the importance of a Surveillance Program to monitor the spread of the plasmid-mediated colistin resistance genes into MDR Gram-negative bacteria. Keywords: Laboratory Surveillance, Escherichia coli, mcr-1, mcr-2, mcr-3, mcr-4, mcr-5, Antimicrobial resistance

The spread of carbapenem-resistant Enterobacteriaceae (CRE) has been enabled by the lack of control measures directed at carriers of multidrug-resistant organisms in healthcare settings. Screening patients for asymptomatic colonization on the one hand, and implementation of contact precautions on the other hand, reduces patient-to-patient transmission. Screening plates represents a relatively low-cost method for isolating CRE from rectal swabs; however, molecular assays have become widely available. This study compared the performance of four commercial molecular platforms in detecting clinically significant carbapenemase genes versus routine screening for CRE. A total of 1015 non-duplicated rectal swabs were cultured on a chromogenic carbapenem-resistant selective medium. All growing Enterobacteriaceae strains were tested for carbapenemase-related genes. The same specimens were processed using the following molecular assays: Allplex™ Entero-DR, Amplidiag® CarbaR + MCR, AusDiagnostics MT CRE EU, and EasyScreen™ ESBL/CPO. The prevalence of Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae detected by swab culture was 2.2%, while organisms producing oxacillinase (OXA)-48 and metallo-β-lactamases were infrequent. The cost of CRE-related infection control precautions, which must be kept in place while waiting for screening results, are significant, so the molecular tests could become cost-competitive, especially when the turnaround time is decreased dramatically. Molecular assays represent a powerful diagnostic tool as they allow the rapid detection of the most clinically relevant carbapenemases.

The Unyvero molecular assay was tested for the clinical resolution of discordant results, evaluating its role in prosthetic joint infection diagnosis.Multiplex PCR was performed on 45 samples from prosthesis treatment (either sonication or dithiothreitol). Analytical performance was compared to that of biofilm culture using Musculoskeletal Infection Society criteria as gold standard.Unyvero and biofilm culture showed similar agreement rates compared to the gold standard (34/43 and 32/43, respectively). Both methods showed six additional identifications compatible with true infection; five positive results from biofilm culture were deemed contaminations.The Unyvero system showed good performances and a significantly shorter turnaround time compared to cultural methods, presenting an added value to PJI diagnosis even when performed following a composite approach.

Background More than one-third of candidemia episodes occur in Internal Medicine Wards (IMWs) but only few studies have focused on this setting and specific data about epidemiology, clinical characteristics and risk factors for mortality are scant. Objective To describe epidemiology and to assess risk factors for in-hospital mortality among patients with candidemia in IMWs. Methods Multicenter retrospective cohort study on patients with candidemia cared for in IMWs of an Italian region (Emilia Romagna) from January 2012 to December 2013. Non survivors were compared with survivors; variables with p ≤ 0.1 at univariate analysis were entered into a multivariate Cox regression model. Results 232 patients were included. Overall candidemia incidence was 2.2 cases/1000 admissions. Candida albicans accounted for 59% of cases. Antifungal treatment was started  72 h from blood cultures in 47%, 27% and 12% of patients, respectively; 13.8% of patients received no antifungal treatment. In-hospital mortality was 40%. At multivariate analysis, chronic-obstructive-pulmonary-disease (HR 2.72, 95%CI 1.66–4.45, pÂ

One of the major concerns in the COVID-19 pandemic is related to the possible transmission in poorly ventilated spaces of SARS-CoV-2 through aerosol microdroplets, which can remain in the air for long periods of time and be transmitted to others over distances '1 m. Cold atmospheric pressure plasmas can represent a promising solution, thanks to their ability in producing a blend of many reactive species, which can inactivate the airborne aerosolized microorganisms. In this study, a dielectric barrier discharge plasma source is used to directly inactivate suitably produced bioaerosols containing Staphylococcus epidermidis or purified SARS-CoV-2 RNA flowing through it. Results show that for low residence times ('0.2 s) in the plasma region a 3.7 log R on bacterial bioaerosol and degradation of viral RNA can be achieved.

OBJECTIVES Sanitary emergency of SARS-CoV-2 is radically changing all the procedures in dental clinics A great number of health-care workers resulted affected by Coronavirus Dentists work closely face-to-face with the patients and this condition greatly increases the risk for Coronavirus infection Normal dental procedures create a tremendous hazard and play a number of risks for contaminations with patient's saliva and exhaled particles All patients may be asymptomatic but Coronavirus-positive and may represent a great risk for virus diffusion in the dental room and among the personnel and/or other patients The principal problem in dental office is constituted by the presence of aerosols and spray droplets created by high-speed water-cooled handpiece, ultrasonic instrument, ultrasonic scalers and dental air-water spray guns The second important problem is connected with the long face-to-face of the operators (dentist and assistants) with patients The airborne fluctuating particles - composed by saliva, mucus, blood, dentin-enamel debris, smear layer, fragments of restorations - can remain floating for a long time in the aerosol tide and are diffused by air vortexes created by high-speed hand-piece MATERIALS AND METHODS The study identified different type of droplets and airborne produced by dental-unit chair (DUC) instruments These droplets and airborne particles are produced by DUC instruments and may be immediately infected by patient mucus and saliva - and in theory plaque and blood - and diffused around as fluctuating particles These particles present a diameter from 200 to 2 microns and may remain in air for long time (minutes and hours) and settle in all the surface around DUC They usually present a ballistic trajectory and may be deviated by turbulences and operator movements Operator masks, glasses, gown etc are all exposed surfaces that may be fast contaminated Also boxes, floor and devices in the dental room may be covered by a layer of droplets Several new devices and new operative techniques were recently developed to try to reduce the spray diffusion and to limit the risk for operator contamination A new designed device is constituted by a double rubber-dam arch with a sliding suction pipe able to uptake spray produced in the mouth and by the nose The device and other types of lip oral cannulas consistently reduce the spray diffusion Other additional innovative procedures for operators are represented by the use of specific waterproof sprayhood to prevent any droplets contact with masks (FFP2/FFP3) and eyes and by Tynek suites to cover all operator's body Finally, all DUC and room disinfection procedures must be evaluated to prevent (and to remove) droplets and particles deposits from the surfaces Additional tailored procedures must be standardized, such as the use of FFP2/3, mounthrinses, disinfection of DUC circuits etc CONCLUSIONS There is the need for all dental offices and dental Clinics to rapidly modify all the clinical procedures in attempt to reduce airborne and spray droplets The close contact of the operators with patient's nose and mouth and the potential presence of virus into mucus and saliva create a tremendous hazard due to the production of DUC spray Again, there is a need for Universities, Dental Clinics and Hospitals to protect students, workers and patients with new tailored operative procedures CLINICAL SIGNIFICANCE In this position paper, it is support the concept that we need major and rapid reorganization of dental office procedures The main concept is to restrict the generation of dental spray and use adequate procedures to prevent diffusion in dental offices New tailored operative procedures are proposed after the evaluation of high risks for airborne and dental spray droplets production and contamination dynamic routes The use of powerful novel suction cannula designed for fast spray/saliva droplets aspiration and the use of innovative sprayhoods and body suites for dental-care worker protections are important procedures to prevent virus transmission in dental office Additional procedures may be standardized to remove droplets from all room surface