Real-time polymerase chain reaction and laser capture microdissection: an efficient combination tool for Chlamydophila pneumoniae DNA quantification and localization of infection in atherosclerotic lesions.

Chlamydophila pneumoniae has been implicated in atherosclerosis, but the role of this obligate intracellular pathogen in the development of the above pathology is still unclear. In particular, its presence and quantitative distribution within lesional areas has not yet been defined. We studied 18 carotid biopsies obtained from patients undergoing endoartherectomy. By laser microdissection (LCM), two different sites (intra-plaque and plaque-adjacent areas) were taken from each lesion, and the presence and quantity of the pathogen DNA were determined by real-time polymerase chain reaction (Real-time PCR). A total of 8 plaques, exclusively, from patients with unstable angina, were positive in real-time PCR. The bacterial DNA was detected in both lesional areas of 3 plaques which contained the highest number of DNA copies (1,900 to 2,200 copy numbers), while C. pneumoniae DNA was detected only in the intra-plaque area of the other 5 positive (500 to 1,600 copy numbers). No C. pneumoniae DNA was found in the other 10 plaques of which 6 were from patients with unstable angina and 4 from stable angina patients. No DNA from Helicobacter pylori or Cytomegalovirus was found in any plaque. This is the first report where both the target lesion and an adjacent reference site were evaluated for the presence of C. pneumoniae DNA by the combination of LCM and Real-time PCR assays. The integration of these two methodologies offer an excellent tool for in situ studies and may help to elucidate the putative role of C. pneumoniae in atherosclerosis

Different pH requirements are associated with divergent inhibitory effects of chloroquine on human and avian influenza A viruses

Chloroquine is a 4-aminoquinoline previously used in malaria therapy and now becoming an emerging investigational antiviral drug due to its broad spectrum of antiviral activities. To explore whether the low pH-dependency of influenza A viruses might affect the antiviral effects of chloroquine at clinically achievable concentrations, we tested the antiviral effects of this drug on selected human and avian viruses belonging to different subtypes and displaying different pH requirements. Results showed a correlation between the responses to chloroquine and NH4Cl, a lysosomotropic agent known to increase the pH of intracellular vesicles. Time-of-addition experiments showed that the inhibitory effect of chloroquine was maximal when the drug had been added at the time of infection and was lost after 2 h post-infection. This timing approximately corresponds to that of virus/cell fusion. Moreover, there was a clear correlation between the EC50 of chloroquine in vitro and the electrostatic potential of the HA subunit (HA2) mediating the virus/cell fusion process. Overall, the present study highlights the critical importance of a host cell factor such as intravesicular pH in determining the anti-influenza activity of chloroquine and other lysosomotropic agents

CARE 1000: randomized controlled trial for the evaluation of the effectiveness of a mHealth app for supporting the first 1000 days of life

Background Recent developments in eHealth and mobile health (mHealth), as well as the introduction of information and communication technology innovations in clinical practice, such as telemedicine, telemonitoring, and remote examinations, are already changing the current scenario and will continue to generate innovations in the coming decades. The widespread use of mobile devices, with an estimated nearly 30 billion devices and more than 325,000 apps worldwide, will provide various opportunities for people to take control of their own health. Already in 2017, most of the apps available were focused on pregnancy support, more than any other medical field. There have been some reported experiences with social media and mHealth that could benefit the promotion of maternal physical and mental health during pregnancy. However, many apps targeting the first 1000 days of a child's life do not consider the continuity between the prenatal and postnatal periods and their joint impact on maternal and child health. The aim of this study is to evaluate the effectiveness of this mHealth app to support women during the first 1000 days (from conception to 24 months of age) and to improve health prevention behaviours such as immunizations during pregnancy, weight gain during pregnancy, abstinence from smoking and alcohol consumption, and adherence to the routine childhood immunization schedule. In addition, the study aims to understand the level of appreciation of this mHealth app as a tool to overcome information and communication gaps between patients and institutions. MethodsConduction of a randomized controlled trial. DiscussionOur results will be relevant for improving this mHealth app to promote health and prevention and to support the first 1000 days of life for both mother and child. Our results will be relevant to the future expansion of such an mHealth app to promote positive health-related outcomes in patients and co-user satisfaction and to support the organization of health services

Fieldable genotyping of Bacillus anthracis and Yersinia pestis based on 25-loci Multi Locus VNTR Analysis

<p>Abstract</p> <p>Background</p> <p>Anthrax and plague are diseases caused by <it>Bacillus anthracis </it>and <it>Yersinia pestis </it>respectively. These bacteria are etiological agents for worldwide zoonotic diseases and are considered among the most feared potential bioterror agents. Strain differentiation is difficult for these microorganisms because of their high intraspecies genome homogeneity. Moreover, fast strain identification and comparison with known genotypes may be crucial for naturally occurring outbreaks versus bioterrorist events discrimination.</p> <p>Results</p> <p>Thirty-nine <it>B. anthracis </it>and ten <it>Y. pestis </it>strains, representative of the species genetic diversity, were genotyped by Agilent 2100 Bioanalyzer using previously described Multiple Locus VNTR Analysis assays (MLVA). Results were compared to previous data obtained by standard genotyping system (capillary electrophoresis on automatic sequencer) and, when necessary, direct amplicon sequencing. A reference comparison table containing actual fragment sizes, sequencer sizes and Agilent sizes was produced.</p> <p>Conclusion</p> <p>In this report an automated DNA electrophoresis apparatus which provides a cheaper alternative compared to capillary electrophoresis approaches was applied for genotyping of <it>B. anthracis </it>and <it>Y. pesti</it>s. This equipment, uses pre-cast gels and provides easy transportation, low maintenance and overall general logistic requirements and costs, is easy to set up and provides rapid analysis. This platform is a candidate for on-site MLVA genotyping of biothreat agents as well as other bacterial pathogens. It is an alternative to the more expensive and demanding capillary electrophoresis methods, and to the less expensive but more time-consuming classical gel electrophoresis approach.</p

Study protocol for a randomised controlled trial to determine the effectiveness of a mHealth application as a family supportive tool in paediatric otolaryngology perioperative process (TONAPP)

Background: Otorhinolaryngology (ORL) surgery is common in children, but hospitalisation, surgery, and home care after discharge are stressful experiences for young patients and their family caregivers. Findings from literature highlight a lack of time in hospitals to support ORL surgery children and their caregivers through the perioperative process, along with the risks of caregivers' autonomous web or social media resources investigation. Therefore, this study aims to evaluate the effectiveness of a mobile health application with content to support ORL patients and their caregivers in the perioperative period to reduce caregiver anxiety and child distress compared to standard care. Methods: An open-label, two-arm randomised control trial design is being adopted. The intervention consists of a mobile health application with content to support ORL patients and their caregivers during the perioperative period. One hundred eighty participants will be enrolled and randomly assigned to the experimental group using the mHealth application or the control group. The control group receives standard information and education about the ORL perioperative period from healthcare providers orally or through brochures. The primary outcome is the difference between the intervention and control groups in preoperative caregiver state anxiety. Secondary outcome measures include children's distress before surgery and family preparation for hospitalisation. Discussion: The results of this study will be critical to the implementation of a new and safe model for the management of care and education in paediatrics. This model can achieve positive organisational and health outcomes by supporting continuity of care and empowering citizens to have informed participation and satisfaction in paediatric health promotion and management. Trial registration: Trial identifier: NCT05460689 registry name: ClinicalTrials.gov. Date of registration: July 15, 2022. Last update posted: February 23, 2023

Genotyping of Bacillus anthracis strains based on automated capillary 25-loci Multiple Locus Variable-Number Tandem Repeats Analysis

BACKGROUND: The genome of Bacillus anthracis, the etiological agent of anthrax, is highly monomorphic which makes differentiation between strains difficult. A Multiple Locus Variable-number tandem repeats (VNTR) Analysis (MLVA) assay based on 20 markers was previously described. It has considerable discrimination power, reproducibility, and low cost, especially since the markers proposed can be typed by agarose-gel electrophoresis. However in an emergency situation, faster genotyping and access to representative databases is necessary. RESULTS: Genotyping of B. anthracis reference strains and isolates from France and Italy was done using a 25 loci MLVA assay combining 21 previously described loci and 4 new ones. DNA was amplified in 4 multiplex PCR reactions and the length of the resulting 25 amplicons was estimated by automated capillary electrophoresis. The results were reproducible and the data were consistent with other gel based methods once differences in mobility patterns were taken into account. Some alleles previously unresolved by agarose gel electrophoresis could be resolved by capillary electrophoresis, thus further increasing the assay resolution. One particular locus, Bams30, is the result of a recombination between a 27 bp tandem repeat and a 9 bp tandem repeat. The analysis of the array illustrates the evolution process of tandem repeats. CONCLUSION: In a crisis situation of suspected bioterrorism, standardization, speed and accuracy, together with the availability of reference typing data are important issues, as illustrated by the 2001 anthrax letters event. In this report we describe an upgrade of the previously published MLVA method for genotyping of B. anthracis and apply the method to the typing of French and Italian B. anthracis strain collections. The increased number of markers studied compared to reports using only 8 loci greatly improves the discrimination power of the technique. An Italian strain belonging to the B branch was described, and two new branches, D and E, are proposed. Owing to the upgrading achieved here, precise genotyping can now be produced either by automated capillary electrophoresis, or by the more accessible but slower and for some markers slightly less accurate agarose gel methodology

Clinical, anamnestic, and sociodemographic predictors of positive SARS-CoV-2 testing in children: A cross sectional study in a tertiary hospital in Italy

ObjectivesWe aimed to identify clinical, anamnestic, and sociodemographic characteristics associated with a positive swab for SARS-CoV2, and to provide a predictive score to identify at risk population in children aged 2-14 years attending school and tested for clinical symptoms of COVID-19.DesignCross sectional study.SettingOutpatient clinic of the IRCCS Burlo Garofolo, a maternal and child health tertiary care hospital and research centre in Italy.Data collection and analysisData were collected through a predefined form, filled out by parents, and gathered information on sociodemographic characteristics, and specific symptoms, which were analysed to determine their association with a positive SARS-CoV-2 swab. The regression coefficients of the variables included in the multivariate analysis were further used in the calculation of a predictive score of the positive or negative test.ResultsBetween September 20th and December 23rd 2020, from 1484 children included in the study, 127 (8.6%) tested positive. In the multivariate analysis, the variables retained by the model were the presence of contact with a cohabiting, non-cohabiting or unspecified symptomatic case (respectively OR 37.2, 95% CI 20.1-68.7; 5.1, 95% CI 2.7-9.6; 15.6, 95% CI 7.3-33.2); female sex (OR 1.49, 95% CI 1.0-2.3); age (6-10 years old: OR 3.2, 95% CI 1.7-6.1 p&lt;0.001; &gt;10 years old: OR 4.8, 95% CI 2.7-8.8 p&lt;0.001); fever (OR 3.9, 95% CI 2.3-6.4); chills (OR 1.9, 95% CI 1.1-3.3); headache (OR 1.45, 95% CI 0.9-2.4); ageusia (OR 1.3, 95% CI 0.54.0); sore throat (OR 0.48, 95% CI 0.3-0.8); earache (OR 0.4, 95% CI 0.1-1.3); rhinorrhoea (OR 0.8, 95% CI 0.5-1.3); and diarrhoea (OR 0.52, 95% CI 0.2-1.1). The predictive score based on these variables generated 93% sensitivity and 99% negative predictive value.ConclusionsThe timely identification of SARS-CoV2 cases among children is useful to reduce the dissemination of the disease and its related burden. The predictive score may be adopted in a public health perspective to rapidly identify at risk children

Health Policy Brief: i pilastri dell'Engagement in Emofilia

About 5000 people in Italy suffer from hemophilia, the most common coagulation disorder. As for other chronic diseases, even in the case of hemophilia, the engagement of the patient is essential: the patients, in fact, must be empowered and helped to become strong partners of the care team and sensitized with respect to their rights and duties for the successful achievement of the goals set by their healthcare path. Hence the initiative to start a new research-intervention project in the field of hemophilia. The study had different phases of research: a first moment inspired by the principles of narrative medicine, aimed at collecting stories and narratives of patients with hemophilia related to the experience of the disease and therapy and expectations of active involvement in the relationship with the clinician. At the same time, among hematologists and patients has been surveyed the experience of therapeutical relationship and communication, to capture the aspects in which they feel effective and the areas of improvement and unmet needs. Subsequently, a workshop dedicated to patients and hematologists was organized to foster mutual awareness between these two targets and the formation of a better communication and relational skills of clinicians. The results of the project formed the basis for a policy brief document, aimed at disseminating recommendations to support better relationship and empathic communication between clinicians and patients

Candida albicans Isolates from the Gut of Critically Ill Patients Respond to Phosphate Limitation by Expressing Filaments and a Lethal Phenotype

Candida albicans is an opportunistic pathogen that proliferates in the intestinal tract of critically ill patients where it continues to be a major cause of infectious-related mortality. The precise cues that shift intestinal C. albicans from its ubiquitous indolent colonizing yeast form to an invasive and lethal filamentous form remain unknown. We have previously shown that severe phosphate depletion develops in the intestinal tract during extreme physiologic stress and plays a major role in shifting intestinal Pseudomonas aeruginosa to express a lethal phenotype via conserved phosphosensory-phosphoregulatory systems. Here we studied whether phosphate dependent virulence expression could be similarly demonstrated for C. albicans. C. albicans isolates from the stool of critically ill patients and laboratory prototype strains (SC5314, BWP17, SN152) were evaluated for morphotype transformation and lethality against C. elegans and mice during exposure to phosphate limitation. Isolates ICU1 and ICU12 were able to filament and kill C. elegans in a phosphate dependent manner. In a mouse model of intestinal phosphate depletion (30% hepatectomy), direct intestinal inoculation of C. albicans caused mortality that was prevented by oral phosphate supplementation. Prototype strains displayed limited responses to phosphate limitation; however, the pho4Δ mutant displayed extensive filamentation during low phosphate conditions compared to its isogenic parent strain SN152, suggesting that mutation in the transcriptional factor Pho4p may sensitize C. albicans to phosphate limitation. Extensive filamentation was also observed in strain ICU12 suggesting that this strain is also sensitized to phosphate limitation. Analysis of the sequence of PHO4 in strain ICU12, its transcriptional response to phosphate limitation, and phosphatase assays confirmed that ICU12 demonstrates a profound response to phosphate limitation. The emergence of strains of C. albicans with marked responsiveness to phosphate limitation may represent a fitness adaptation to the complex and nutrient scarce environment typical of the gut of a critically ill patient