Persistence of SARS-CoV-2 infection and viral intra- and inter-host evolution in COVID-19 hospitalized patients.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) persistence in COVID-19 patients could play a key role in the emergence of variants of concern. The rapid intra-host evolution of SARS-CoV-2 may result in an increased transmissibility, immune and therapeutic escape which could be a direct consequence of COVID-19 epidemic currents. In this context, a longitudinal retrospective study on eight consecutive COVID-19 patients with persistent SARS-CoV-2 infection, from January 2022 to March 2023, was conducted. To characterize the intra- and inter-host viral evolution, whole genome sequencing and phylogenetic analysis were performed on nasopharyngeal samples collected at different time points. Phylogenetic reconstruction revealed an accelerated SARS-CoV-2 intra-host evolution and emergence of antigenically divergent variants. The Bayesian inference and principal coordinate analysis analysis showed a host-based genomic structuring among antigenically divergent variants, that might reflect the positive effect of containment practices, within the critical hospital area. All longitudinal antigenically divergent isolates shared a wide range of amino acidic (aa) changes, particularly in the Spike (S) glycoprotein, that increased viral transmissibility (K417N, S477N, N501Y and Q498R), enhanced infectivity (R346T, S373P, R408S, T478K, Q498R, Y505H, D614G, H655Y, N679K and P681H), caused host immune escape (S371L, S375F, T376A, K417N, and K444T/R) and displayed partial or complete resistance to treatments (G339D, R346K/T, S371F/L, S375F, T376A, D405N, N440K, G446S, N460K, E484A, F486V, Q493R, G496S and Q498R). These results suggest that multiple novel variants which emerge in the patient during persistent infection, might spread to another individual and continue to evolve. A pro-active genomic surveillance of persistent SARS-CoV-2 infected patients is recommended to identify genetically divergent lineages before their diffusion.

A systematic review on antibiotic therapy of cutaneous bacillary angiomatosis not related to major immunocompromising conditions: from pathogenesis to treatment.

BACKGROUND: Cutaneous bacillary angiomatosis (cBA) is a vascular proliferative disorder due to Bartonella spp. that mostly affects people living with HIV (PLWH), transplanted patients and those taking immunosuppressive drugs. Since cBA is mostly related to these major immunocompromising conditions (i.e., T-cell count impairment), it is considered rare in relatively immunocompetent patients and could be underdiagnosed in them. Moreover, antimicrobial treatment in this population has not been previously investigated. METHODS: We searched the databases PubMed, Google Scholar, Scopus, OpenAIRE and ScienceDirect by screening articles whose title included the keywords "bacillary" AND "angiomatosis" and included case reports about patients not suffering from major immunocompromising conditions to provide insights about antibiotic treatments and their duration. RESULTS: Twenty-two cases of cBA not related to major immunocompromising conditions were retrieved. Antibiotic treatment duration was shorter in patients with single cBA lesion than in patients with multiple lesions, including in most cases macrolides and tetracyclines. CONCLUSIONS: cBA is an emerging manifestation of Bartonella spp. infection in people not suffering from major immunocompromising conditions. Until evidence-based guidelines are available, molecular tests together with severity and extension of the disease can be useful to personalize the type of treatment and its duration.

Changing and evolution of influenza virus: is it a trivial flu?

BACKGROUND: Influenza viruses are etiological agents which cause contagious respiratory, seasonal epidemics and, for Influenza A subtypes, pandemics. The clinical picture of Influenza has undergone continuous change over the years, due to intrinsic viral evolution as well as "reassortment" of its genomic segments. The history of Influenza highlights its ability to adapt and to rapidly evolve, without specific circumstances. This reflects the complexity of this pathology and poses the fundamental question about its assumption as a "common illness" and its impact on public health. SUMMARY: The global influenza epidemics and pandemics claimed millions of deaths, leaving an indelible mark on public health, and showing the need for a better comprehension of the influenza virus. The clear understanding of genetic variations during the Influenza seasonal epidemics is a crucial point for developing effective strategies for prevention, treatment, and vaccine design. The recent advance in Next Generation Sequencing approaches, model systems to virus culture and bioinformatics pipeline played a key role in the rapid characterization of circulating Influenza strains. In particular, the increase of computational power allowed to perform complex tasks in healthcare setting through Machine Learning (ML) algorithms, which analyze different variables, such as medical and laboratory outputs, to optimize medical research and to improve public health systems. The early detection of emerging and re-emerging pathogens is of matter importance to prevent next pandemics. KEY MESSAGES: The perception of influenza as a "trivial flu" or a more serious public health concern is a subject of ongoing debate, reflecting the multifaceted nature of this infectious disease. The variability in the severity of influenza shed the light on the unpredictability of the viral characteristics, coupled with the challenges in accurately predicting circulating strains. This adds complexity to the public health burden of Influenza and highlights the need of targeted interventions.

Gut Microbiota Profile Changes in Patients with Inflammatory Bowel Disease and Non-Alcoholic Fatty Liver Disease: A Metagenomic Study.

Gut microbiota imbalances have a significant role in the pathogenesis of Inflammatory Bowel Disease (IBD) and Non-Alcoholic Fatty Liver Disease (NAFLD). Herein, we compared gut microbial composition in patients diagnosed with either IBD or NAFLD or a combination of both. Seventy-four participants were stratified into four groups: IBD-NAFLD, IBD-only, NAFLD-only patients, and healthy controls (CTRLs). The 16S rRNA was sequenced by Next-Generation Sequencing. Bioinformatics and statistical analysis were performed. Bacterial α-diversity showed a significant lower value when the IBD-only group was compared to the other groups and particularly against the IBD-NAFLD group. ß-diversity also showed a significant difference among groups. The higher Bacteroidetes/Firmicutes ratio was found only when comparing IBD groups and CTRLs. Comparing the IBD-only group with the IBD-NAFLD group, a decrease in differential abundance of Subdoligranulum, Parabacteroides, and Fusicatenibacter was found. Comparing the NAFLD-only with the IBD-NAFLD groups, there was a higher abundance of Alistipes, Odoribacter, Sutterella, and Lachnospira. An inverse relationship in the comparison between the IBD-only group and the other groups was shown. For the first time, the singularity of the gut microbial composition in IBD and NAFLD patients has been shown, implying a potential microbial signature mainly influenced by gut inflammation.

SARS CoV-2 spike protein-guided exosome isolation facilitates detection of potential miRNA biomarkers in COVID-19 infections.

OBJECTIVES: Nearly three years into the pandemic, SARS-CoV-2 infections are occurring in vaccinated and naturally infected populations. While humoral and cellular responses in COVID-19 are being characterized, novel immune biomarkers also being identified. Recently, an increase in angiotensin-converting enzyme 2 expressing (aka, ACE2 positive) circulating exosomes (ExoACE2) were identified in the plasma of COVID-19 patients (El-Shennawy et al.). In this pilot study, we describe a method to characterize the exosome-associated microRNA (exo-miRNA) signature in ACE2-positive and ACE2-negative exosomal populations (non-ExoACE2). METHODS: We performed a sorting protocol using the recombinant biotin-conjugated SARS CoV-2 spike protein containing the receptor binding domain (RBD) on plasma samples from six patients. Following purification, exo-miRNA were characterized for ACE2-positive and ACE2-negative exosome subpopulations by RT-PCR. RESULTS: We identified differential expression of several miRNA. Specifically let-7g-5p and hsa-miR-4454+miR-7975 were upregulated, while hsa-miR-208a-3p and has-miR-323-3p were downregulated in ExoACE2 vs. non-ExoACE2. CONCLUSIONS: The SARS CoV-2 spike-protein guided exosome isolation permits isolation of ExoACE2 exosomes. Such purification facilitates detailed characterization of potential biomarkers (e.g. exo-miRNA) for COVID-19 patients. This method could be used for future studies to further the understanding mechanisms of host response against SARS CoV-2.

The Role of Gut Microbiota in the Clinical Outcome of Septic Patients: State of the Art and Future Perspectives.

Sepsis is a life-threatening multiple-organ dysfunction caused by a dysregulated host response to infection, with high mortality worldwide; 11 million deaths per year are attributable to sepsis in high-income countries. Several research groups have reported that septic patients display a dysbiotic gut microbiota, often related to high mortality. Based on current knowledge, in this narrative review, we revised original articles, clinical trials, and pilot studies to evaluate the beneficial effect of gut microbiota manipulation in clinical practice, starting from an early diagnosis of sepsis and an in-depth analysis of gut microbiota.

Lessons learned and implications of early therapies for coronavirus disease in a territorial service centre in the Calabria region: a retrospective study.

BACKGROUND: Monoclonal antibodies (mAbs) and antivirals have been approved for early therapy of coronavirus disease (COVID-19), however, in the real-life setting, there are difficulties to prescribe these therapies within few days from symptom onset as recommended, and effectiveness of combined use of these drugs have been hypothesised in most-at-risk patients (such as those immunocompromised) but data supporting this strategy are limited. METHODS: We describe the real-life experience of SARS-CoV-2 antivirals and/or monoclonal antibodies (mAbs) and focus on the hospitalisation rate due to the progression of COVID-19. Clinical results obtained through our risk-stratification algorithm and benefits achieved through a strategic proximity territorial centre are provided. We also report a case series with an in-depth evaluation of SARS-CoV-2 genome in relationship with treatment strategy and clinical evolution of patients. RESULTS: Two hundred eighty-eight patients were analysed; 94/288 (32.6%) patients were treated with mAb monotherapy, 171/288 (59.4%) patients were treated with antivirals, and 23/288 (8%) patients received both mAbs and one antiviral drug. Haematological malignancies were more frequent in patients treated with combination therapy than in the other groups (p = 0.0003). There was a substantial increase in the number of treated patients since the opening of the centre dedicated to early therapies for COVID-19. The provided disease-management and treatment appeared to be effective since 98.6% patients recovered without hospital admission. Moreover, combination therapy with mAbs and antivirals seemed successful because all patients admitted to the hospital for COVID-19 did not receive such therapies, while none of the most-at-risk patients treated with combination therapy were hospitalized or reported adverse events. CONCLUSIONS: A low rate of COVID-19 progression requiring hospital admission was observed in patients included in this study. The dedicated COVID-19 proximity territorial service appeared to strengthen the regional sanitary system, avoiding the overwhelming of other services. Importantly, our results also support early combination therapy: it is possible that this strategy reduces the emergence of escape mutants of SARS-CoV-2, thereby increasing efficacy of early treatment, especially in immunocompromised individuals.

Role of the T2Dx magnetic resonance assay in patients with suspected bloodstream infection: a single-centre real-world experience.

BACKGROUND: T2Dx was approved by the US Food and Drug Administration for the rapid detection of a modified panel of ESKAPE bacterial species or Candida spp. causing bloodstream infection (BSI). PATIENTS AND METHODS: We performed a retrospective, observational study from January 1, 2018 to December 31, 2019 of all hospitalised patients with suspected BSI who underwent assessment using T2Dx in addition to standard blood culture (BC). T2-positive patients (cases) were compared to a matched group of patients with BSI documented only by BC (1:2 ratio) to investigate the possible impact of T2Dx on the appropriateness of empirical antimicrobial therapy and 21-day mortality. RESULTS: In total, 78 T2Dx-analysed samples (49 patients) were analysed. The T2Dx assay result was positive for18 patients and negative for 31 patients. The concordance rates of the T2Bacteria Panel and T2Candida Panel results with those of standard BC were 74.4% and 91.4%, respectively. In the matched analysis, inappropriate empiric antimicrobial therapy administration was significantly less frequent in cases than in comparators (5.5% vs. 38.8%). The 21-day mortality rate was twofold lower in cases than in comparators (22.2% vs. 44.4%), although the difference was not significant. No other analysed variables were significantly different between the two groups. CONCLUSIONS: This study illustrated that T2Dx might be associated with an increase in the appropriateness of empiric antimicrobial therapy in patients with BSI. Further studies are needed to evaluate whether the T2Dx assay can improve patient outcomes.

Identification of pmrB mutations as putative mechanism for colistin resistance in A. baumannii strains isolated after in vivo colistin exposure.

Colistin resistance among extensively-resistant Acinetobacter baumannii isolates is a serious health-care problem. Alterations in PmrA-PmrB two-component system have been associated with resistance to colistin. We investigated three pairs of colistin-susceptible and colistin-resistant A. baumannii, sequentially isolated from three patients before and after colistin treatment, respectively. The pmrA and pmrB genes were sequenced by Sanger method. Amino acidic positions and their effect on protein were predicted by InterPro and PROVEAN tools. Expression of pmrA, pmrB and pmrC genes was assessed by semi-quantitative reverse transcription-PCR (qRT-PCR). We found three different nonsynonymous substitutions P233T, E301G and L168K in pmrB coding region, each one in a different colistin resistance strain. The E301G and L168K substitutions represent novel mutations in pmrB, not previously described. Relative expression of pmrA, pmrB and pmrC mRNA increased in all colistin resistant strains. In our study, pmrB substitutions were associated with pmrC over-expression and colistin resistance. Further studies are necessary to understand their impact on modification of lipid A components.

Clinical outcomes of patients treated with intravenous zanamivir for severe influenza A(H1N1)pdm09 infection: a case report series.

BACKGROUND: Intravenous (IV) zanamivir could be a suitable alternative for the treatment of severe influenza A(H1N1)pdm09 infection in patients who are unable to take oral or inhaled medication, for example, those on mechanical ventilation and extracorporeal membrane oxygenation (ECMO). However, data on the clinical outcomes of such patients is limited. CASE PRESENTATION: We report the clinical outcomes of four patients who were admitted at the intensive care unit during the 2017-2018 influenza season with severe sepsis (SOFA score > 11) and acute respiratory distress syndrome requiring ECMO and mechanical ventilation. Two patients were immune-compromised. The A(H1N1)pdm09 genome was confirmed by polymerase chain reaction (PCR) on nasopharyngeal specimen swabs prior to administration of IV zanamivir at a dose of 600 mg twice daily. Weekly qualitative PCR analysis was done to monitor viral clearance, with zanamivir treatment being discontinued upon receipt of negative results. In addition, the patients were managed for concomitant multidrug-resistant bacterial infections, with infection resolution confirmed with blood cultures. The median time for zanamivir treatment was 10 days (IQR 10-17). The clinical outcome was favourable with all four patients surviving and improving clinically. All four patients achieved viral clearance of A(H1N1)pdm09 genome, and resolution of multidrug-resistant bacterial infections. CONCLUSIONS: IV zanamivir could be a good therapeutic option in patients with severe influenza A(H1N1)pdm09 infection who are unable to take oral or aerosolised antiviral medication. We recommend prospective randomized control trials to support this hypothesis.

[A French recovery-oriented mental health service based on housing.] / Un service psychiatrique français orienté vers le rétablissement par le logement.

INTRODUCTION: The article aims at examining the various forms of mental healthcare services’ organization based on housing, in a context influenced by the innovative experiments of the deinstitutionalization struggles of the 1970’s. A Housing Service, having operated for decades to facilitate social integration, is currently focusing on promoting patients’ autonomy in their own apartment. An orientation to recovery has been adopted in the assistance of more autonomous patients who have not suffered any long-term hospitalization: they are considered as contractual users of the service. METHOD: An ethnographic approach applied to a case study, combined with the tools of the sociology of engagement regimes, aims at analyzing how an instance of non-compliance affects the capacity-oriented organization of the service, through the apartment. RESULTS: In the case that we have observed, the user’s refusal triggers an assessment procedure and a series of tests implied by the capacity-support approach of the service which eventually leads to the re-orientation of the assistance from the apartment to a medical facility. CONCLUSION: The medical objective has been prioritized over social integration. We explore alternative ways to maintain the apartment as a therapeutic and social integration tool.

Increase of Vascular Endothelial Growth Factor and Decrease of MCP-1 and Some Updated Epidemiology Aspects of Cystic Echinococcosis Human Cases in Calabria Region.

We aim to investigate some of the pathogenetic mediators of the human echinococcosis and to obtain updated epidemiological findings on cases of echinococcosis in Calabria, Southern Italy. Echinococcosis diagnosis was based on imaging, serological investigations, and molecular assay. Indeed, real-time PCR indicated the presence of G2/G3 genotypes of Echinococcus granulosus complex. Regarding pathogenesis, a relevant novel tool of immune depression should be deemed the reduced level of serum MCP-1. Also, we found a previously unreported VEGF, possibly associated with neovascularization requested by the parasite cyst metabolism. Cytokine profiles suggest a bias of the immunity toward Th2 and Treg responses. Nitric oxide levels exhibited a significant decrease one week after therapy versus basal level measured before surgery and/or chemotherapy. An increase of serum total IgE class and IgG4 subclass was found in Echinococcus-positive patients versus controls. Our data demonstrated an endemic spreading, at least in the province of Catanzaro and neighboring Calabria territories, for such parasitosis with the novel issue of the number of female overcoming male cases. In conclusion, the novel findings of this study were the increased VEGF and the reduced serum MCP-1 in the studied cases, as well as the number of Echinococcus-infected females overcoming the infected males.

Typing of Ochrobactrum anthropi clinical isolates using automated repetitive extragenic palindromic-polymerase chain reaction DNA fingerprinting and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

BACKGROUND: Ochrobactrum anthropi (O. anthropi), is a non-fermenting gram-negative bacillus usually found in the environment. Nevertheless, during the past decade it has been identified as pathogenic to immunocompromised patients. In this study, we assessed the usefulness of the automated repetitive extragenic palindromic-polymerase chain reaction (rep-PCR-based DiversiLab™ system, bioMèrieux, France) and of matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF MS) for typing of twentythree O. anthropi clinical isolates that we found over a four-months period (from April 2011 to August 2011) in bacteriemic patients admitted in the same operative unit of our hospital. Pulsed-field gel electrophoresis (PFGE), commonly accepted as the gold standard technique for typing, was also used. Analysis was carried out using the Pearson correlation coefficient to determine the distance matrice and the unweighted pair group method with arithmetic mean (UPGMA) to generate dendogram. RESULTS: Rep-PCR analysis identified four different patterns: three that clustered together with 97% or more pattern similarity, and one whose members showed < 95% pattern similarity. Interestingly, strains isolated later (from 11/06/2011 to 24/08/2011) displayed a pattern with 99% similarity. MALDI-TOF MS evaluation clustered the twentythree strains of O. anthropi into a single group containing four distinct subgroups, each comprising the majority of strains clustering below 5 distance levels, indicating a high similarity between the isolates. CONCLUSIONS: Our results indicate that these isolates are clonally-related and the methods used afforded a valuable contribution to the epidemiology, prevention and control of the infections caused by this pathogen.

Challenging clinical cases in HCV infection.

We present clinical cases, which underline some difficulties in diagnosis and treatment of hepatitis C virus (HCV) infection. Case report #1 shows a patient who avoided clinical follow-up for HCV until the development of hepatocellular carcinoma. In this patient, non-invasive procedures did not allow to make a differential diagnosis between hydatidosis and hepatocellular carcinoma but diagnosis was only made with liver biopsy.

Present, old and future strategies for anti-HCV treatment in patients infected by genotype-1: estimation of the drug costs in the Calabria Region in the era of the directly acting antivirals.

BACKGROUND: In Italy, anti-HCV drugs are provided free of charge by the National Health System. Since 2011, three drug regimens including a directly acting antiviral (DAA) are considered the gold standard for HCV treatment. However, these drugs add a significant cost (roughly €26,000) to the combination of pegylated-interferon-α/ribavirin (PEG-IFN/RBV), which before DAA represented the unique treatment. To provide the National Health System potential useful information, we estimated costs to provide anti-HCV drugs to treat a population experienced for PEG-INF/RBV. METHODS: Genotype 1 HCV mono-infected or HIV/HCV co-infected individuals who were treated with PEG-IFN/RBV between 2008 and 2013 were included. The cost to treat these patients with PEG-IFN/RBV was calculated (cost 1). We also estimated costs if we had to treat these patients with a lead-in period of PEG-INF/RBV followed by PEG-IFN/RBV and a DAA in naïves (cost 2), in addition to cost 1 plus the estimated cost to re-treat with PEG-IFN/RBV and a DAA patients who had a relapse or a non response (cost 3). Moreover, all costs were normalized by SVR. Rates of foreseen response with DAA were obtained from literature data. RESULTS: The overall study population consisted of 104 patients. The rate of sustained virological response (SVR) was 55%, while it was estimated that SVR would be obtained in 75% of patients with a lead-in period with PEG-IFN/RBV followed by a DAA combination, and in 78% if this treatment is used to re-treat experienced patients with a DAA. Drug costs associated with these treatments were: €1,214,283 for cost 1, €3,474,977 for cost 2 and €3,002,095 for cost 3. Costs per SVR achieved were: €22,284 for cost 1, €44,643 for cost 2 and €38,322 for cost 3. CONCLUSIONS: Treatments including DAAs achieve a SVR in more patients than PEG-IFN/RBV but they cost around three times more than PEG-IFN/RBV alone regimens. Also, cost per SVR is almost twofold greater than PEG-IFN/RBV regimens. Therefore, it is mandatory to implement use of DAA in clinical practice, but the National Health System should allocate adequate resources to provide drugs, which challenges sustainability. Cost reduction for anti-HCV drugs should be pursued.

Update on epidemiology of HCV in Italy: focus on the Calabria Region.

The epidemiological profile of HCV infection is evolving in Europe, as well as in Italy. We have previously showed genotype distributions and their dynamics in 2,153 HCV RNA positive patients living in Calabria, Southern Italy, over 11 years. In this study, we extend and update this information by evaluating a hospital-based cohort of 945 HCV RNA positive patients attending five hospitals in the Calabria Region from January 2011 to August 2013. We assessed rates of HCV genotypes according to age and gender and the dynamics of HCV genotype distribution over the 3-year period studied. Data showed that genotype 1b is the most prevalent, followed by subtypes 2a/2c and genotype 3. Genotype 4 exhibited an increase between 2011 and 2013. Also, we found a significant decrease in the median age of subjects infected with HCV genotype 3 and 4 during the period studied. Since HCV genotypes are important in epidemiology, pathogenesis and response to antiviral therapy, a continuous epidemiological surveillance is needed.