Implementation of an antimicrobial stewardship program in the Vascular Surgery ward of a university tertiary care hospital in Pavia, Northern Italy.

PURPOSE: The commitment of multidisciplinary teams in antimicrobial stewardship programs (ASPs) is often inadequately considered, especially in surgical wards. We wanted to evaluate clinical, microbiological, and pharmacological outcomes before and after the implementation of an ASP in the Vascular Surgery ward of Fondazione IRCCS Policlinico San Matteo, a tertiary care hospital in Pavia, Italy. METHODS: This was a quasi-experimental quality-improvement study. The antimicrobial stewardship activity was conducted twice a week for 12 months and consisted of both prospective audit and feedback of all the ongoing antimicrobial prescriptions by the infectious diseases' consultants and educational meetings for the healthcare workers of the Vascular Surgery ward. For comparison between the study periods, Student t test (Mann-Whitney test for skewed distributions) was used for quantitative variables (ANOVA or Kruskall-Wallis for > 2 groups respectively), and Pearson's chi-squared test (Fisher exact test where appropriate) for categorical variables. 2-tailed tests were used. P-value significance cut-off was 0.05. RESULTS: During the 12-month intervention period, among a total number of 698 patients, 186 prescriptions were revised, mostly leading to de-escalating an ongoing antimicrobial therapy (39, 20.97%). A statistically significant reduction in isolates of carbapenem-resistant Pseudomonas aeruginosa (p-value 0.003) and the absence of Clostridioides difficile infections were reported. No statistically significant changes were observed in terms of length of stay and all-cause in-hospital mortality. A significant decrease in the administration of carbapenems (p-value 0.01), daptomycin (p-value < 0.01) and linezolid (p-value 0.43) was registered. A significant reduction in antimicrobial costs was also observed. CONCLUSIONS: The implementation of a 12-month ASP brought significant clinical and economic results, highlighting the benefits of a multidisciplinary teamwork.

The impact of HIV infection and men who have sex with men status on hepatitis A infection: The experience of two tertiary centres in Northern Italy during the 2017 outbreak and in the 2009-2016 period.

Hepatitis A is a self-limiting infection representing the most common cause of viral hepatitis worldwide. Despite being a low incidence region, in the European Union, an increasing number of cases have been reported since summer 2016, resulting in a large outbreak in 2017, involving mainly men who have sex with men (MSM). Some reports described a different clinical course of hepatitis A virus in patients infected by human immunodeficiency virus (HIV) or MSM. We consecutively collected all the hospitalized cases of hepatitis A referred to two tertiary centres in Northern Italy in 2017 and retrospectively analysed the electronic records of the 2009-2016 period (pre-2017). We evaluated demographics data, risk factors, comorbidities and laboratory results to see whether MSM status or HIV infection influenced the disease. Overall, 117 cases were identified in 2017:107 (91%) were male, 78 reported themselves as MSM (66%) and 17 (14.5%) were infected by HIV. For the pre-2017 period, 48 cases were reported: 29 (60%) were male and 3 (6.2%) were infected by HIV. After stratification for HIV infection, MSM status and occurrence period, no differences were found in aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transpeptidase; bilirubin, alkaline phosphatase and bilirubin values, hospitalization length, HIV viral load and CD4 + cells count. HIV-positive patients presented a higher number of patients with INR > 1.5 at admission. MSM status and HIV infection did not affect neither the clinical course nor the severity of hepatitis A.

Epidemiology of Mycobacterium tuberculosis infection in Pavia province, Lombardy, Northern Italy, 1998-2013.

This study investigated the epidemiology of tuberculosis in the last 16 years in the province of Pavia, Lombardy, Northern Italy. The objective was to evaluate the clinical pattern of tuberculosis in immigrant groups compared with Italians in an observational retrospective study conducted from 1998 to 2013. In all, 615 tuberculosis cases were admitted, 354 males (57.3%), median age 47-years, 425 (69.1%) Italian-born patients, 190 (30.9%) immigrants. The ratio between the immigrant group and the Italian-born group of patients increased from 1.7% to 54.5% in the study period (p=0.001). HIV was the most common comorbidity, affecting 48 patients (7.8%), followed by diabetes in 35 (5.7%) and COPD in 30 (4.9%). The overall admission-associated mortality was 5.5%. Italian-born patients were older than non-Italian born subjects and had at least one comorbidity, 162 (38.1%) and 22 (11.6%), respectively (p<0,0001). Mortality was increased among Italian-born compared with non-Italian-born patients (7.3% versus 1.6%, p=0.004). No significant variation in extra-pulmonary tuberculosis (EPTB) prevalence occurred. Considering specific form of EPTB, HIV infection was associated with an increased risk of EPTB (RR 2.02, 95%CI 1.09-3.74, p=0.026). There was a high risk of tuberculosis among immigrants, whereas a decreasing trend was consistently observed among Italian-born patients. Italian-born patients show a higher tuberculosis-associated mortality risk due to older age and comorbidities.

The molecular basis of tumor metastasis and current approaches to decode targeted migration-promoting events in pediatric neuroblastoma.

Cell motility is a crucial biological process that plays a critical role in the development of multicellular organisms and is essential for tissue formation and regeneration. However, uncontrolled cell motility can lead to the development of various diseases, including neoplasms. In this review, we discuss recent advances in the discovery of regulatory mechanisms underlying the metastatic spread of neuroblastoma, a solid pediatric tumor that originates in the embryonic migratory cells of the neural crest. The highly motile phenotype of metastatic neuroblastoma cells requires targeting of intracellular and extracellular processes, that, if affected, would be helpful for the treatment of high-risk patients with neuroblastoma, for whom current therapies remain inadequate. Development of new potentially migration-inhibiting compounds and standardized preclinical approaches for the selection of anti-metastatic drugs in neuroblastoma will also be discussed.

Post-acute coronavirus disease 2019 (COVID 19) syndrome: HLH and cholangiopathy in a lung transplant recipient.

Background: Sars-CoV2 can cause severe pneumonia and acute respiratory distress syndrome (ARDS). In COVID-19-associated respiratory failure, lung transplantation might be an option (Bharat A). Case report: A previously healthy 63-year-old man with a nasopharyngeal swab positive for SarsCoV2 and radiological evidence of interstitial lung consolidations developed acute respiratory distress that required intubation and veno-venous extracorporeal membrane oxygenation support (VV ECMO). Because of no recovery of his lung function, he underwent a bilateral lung transplant. ICU stay was complicated by several episodes of bacterial superinfections and an increase of liver function tests (LFTs). Afterward, he faced a progressive clinical worsening associated to severe anemia, further rise of indices of cholestasis, hypertriglyceridemia and hyperferritinemia. Bone marrow smear showed a picture compatible with haemophagocytic lymphohistocytosis (HLH) and first and second line therapy were started. In addition, a transjugular hepatic biopsy was performed with histopathological evidence of portal and periportal fibrosis, compatible with Covid 19-related cholangiopathy. During the hospital stay, he developed several MDR opportunistic infections. The patient died few months later from multiorgan failure secondary to septic shock. A post-mortem confirmed a diagnosis of cholangiopathy, and medullary erythro-haemophagocytosis. Conclusion: Post Covid19 syndrome is a clinical entity that includes novel and old sequelae following recovery from Sars-CoV2 infections. Early identification of these diseases is crucial for adequate management and might influence the long term prognosis of these patients.

Spectrally Tunable Förster Resonance Energy Transfer-Based Biosensors Using Organic Dye Grafting.

Biosensors based on Förster resonance energy transfer (FRET) have revolutionized cellular biology by allowing the direct measurement of biochemical processes in situ. Many genetically encoded sensors make use of fluorescent proteins that are limited in spectral versatility and that allow few ways to change the spectral properties once the construct has been created. In this work, we developed genetically encoded FRET biosensors based on the chemigenetic SNAP and HaloTag domains combined with matching organic fluorophores. We found that the resulting constructs can display comparable responses, kinetics, and reversibility compared to their fluorescent protein-based ancestors, but with the added advantage of spectral versatility, including the availability of red-shifted dye pairs. However, we also find that the introduction of these tags can alter the sensor readout, showing that careful validation is required before applying such constructs in practice. Overall, our approach delivers an innovative methodology that can readily expand the spectral variety and versatility of FRET-based biosensors.

Modifying PCDH19 levels affects cortical interneuron migration.

PCDH19 is a transmembrane protein and member of the protocadherin family. It is encoded by the X-chromosome and more than 200 mutations have been linked to the neurodevelopmental PCDH-clustering epilepsy (PCDH19-CE) syndrome. A disturbed cell-cell contact that arises when random X-inactivation creates mosaic absence of PCDH19 has been proposed to cause the syndrome. Several studies have shown roles for PCDH19 in neuronal proliferation, migration, and synapse function, yet most of them have focused on cortical and hippocampal neurons. As epilepsy can also be caused by impaired interneuron migration, we studied the role of PCDH19 in cortical interneurons during embryogenesis. We show that cortical interneuron migration is affected by altering PCDH19 dosage by means of overexpression in brain slices and medial ganglionic eminence (MGE) explants. We also detect subtle defects when PCDH19 expression was reduced in MGE explants, suggesting that the dosage of PCDH19 is important for proper interneuron migration. We confirm this finding in vivo by showing a mild reduction in interneuron migration in heterozygote, but not in homozygote PCDH19 knockout animals. In addition, we provide evidence that subdomains of PCDH19 have a different impact on cell survival and interneuron migration. Intriguingly, we also observed domain-dependent differences in migration of the non-targeted cell population in explants, demonstrating a non-cell-autonomous effect of PCDH19 dosage changes. Overall, our findings suggest new roles for the extracellular and cytoplasmic domains of PCDH19 and support that cortical interneuron migration is dependent on balanced PCDH19 dosage.

Immunity to SARS-CoV-2 up to 15 months after infection.

Information concerning the longevity of immunity to SARS-CoV-2 following natural infection may have considerable implications for durability of immunity induced by vaccines. Here, we monitored the SARS-CoV-2 specific immune response in COVID-19 patients followed up to 15 months after symptoms onset. Following a peak at day 15-28 postinfection, the IgG antibody response and plasma neutralizing titers gradually decreased over time but stabilized after 6 months. Compared to G614, plasma neutralizing titers were more than 8-fold lower against variants Beta, Gamma, and Delta. SARS-CoV-2-specific memory B and T cells persisted in the majority of patients up to 15 months although a significant decrease in specific T cells, but not B cells, was observed between 6 and 15 months. Antiviral specific immunity, especially memory B cells in COVID-19 convalescent patients, is long-lasting, but some variants of concern may at least partially escape the neutralizing activity of plasma antibodies.

SARS-CoV-2 specific T-cell immunity in COVID-19 convalescent patients and unexposed controls measured by ex vivo ELISpot assay.

OBJECTIVES: SARS-CoV-2 T-cell response characterization represents a crucial issue for defining the role of immune protection against COVID-19. The aim of the study was to assess the SARS-CoV-2 T-cell response in a cohort of COVID-19 convalescent patients and in a group of unexposed subjects. METHODS: SARS-CoV-2 T-cell response was quantified from peripheral blood mononuclear cells (PBMCs) of 87 COVID-19 convalescent subjects (range 7-239 days after symptom onset) and 33 unexposed donors by ex vivo ELISpot assay. Follow-up of SARS-CoV-2 T-cell response was performed in ten subjects up to 12 months after symptom onset. The role of SARS-CoV-2 specific CD4 and CD8 T cells was characterized in a group of COVID-19 convalescent subjects. Moreover, neutralizing antibodies were determined in serum samples. RESULTS: In 14/33 (42.4%) unexposed donors and 85/87 (97.7%) COVID-19 convalescent subjects a positive result for at least one SARS-CoV-2 antigen was observed. A positive response was observed up to 12 months after COVID-19 infection (median 246 days after symptom onset; range 118-362 days). Of note, SARS-CoV-2 T-cell response seems to be mainly mediated by CD4 T cells. A weak positive correlation was observed between Spike-specific T-cell response and neutralizing antibody titre (p 0.0028; r2 = 0.2891) and positive SARS-CoV-2 T-cell response was observed in 8/9 (88.9%) COVID-19 convalescent subjects with undetectable SARS-CoV-2 neutralizing antibodies. DISCUSSION: Cross-reactive SARS-CoV-2 T-cell response in uninfected patients may be due to previous infections with other common coronaviruses. Our data suggest that long-term SARS-CoV-2 T-cell response might accompany a waning humoral response.

Unique immunological profile in patients with COVID-19.

The relationship between severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and host immunity is poorly understood. We performed an extensive analysis of immune responses in 32 patients with severe COVID-19, some of whom succumbed. A control population of healthy subjects was included. Patients with COVID-19 had an altered distribution of peripheral blood lymphocytes, with an increased proportion of mature natural killer (NK) cells and low T-cell numbers. NK cells and CD8+ T cells overexpressed T-cell immunoglobulin and mucin domain-3 (TIM-3) and CD69. NK cell exhaustion was attested by increased frequencies of programmed cell death protein 1 (PD-1) positive cells and reduced frequencies of natural killer group 2 member D (NKG2D)-, DNAX accessory molecule-1 (DNAM-1)- and sialic acid-binding Ig-like lectin 7 (Siglec-7)-expressing NK cells, associated with a reduced ability to secrete interferon (IFN)γ. Patients with poor outcome showed a contraction of immature CD56bright and an expansion of mature CD57+ FcεRIγneg adaptive NK cells compared to survivors. Increased serum levels of IL-6 were also more frequently identified in deceased patients compared to survivors. Of note, monocytes secreted abundant quantities of IL-6, IL-8, and IL-1ß which persisted at lower levels several weeks after recovery with concomitant normalization of CD69, PD-1 and TIM-3 expression and restoration of CD8+ T cell numbers. A hyperactivated/exhausted immune response dominate in severe SARS-CoV-2 infection, probably driven by an uncontrolled secretion of inflammatory cytokines by monocytes. These findings unveil a unique immunological profile in COVID-19 patients that will help to design effective stage-specific treatments for this potentially deadly disease.

Persistence of SARS-CoV-2-specific B and T cell responses in convalescent COVID-19 patients 6-8 months after the infection.

BACKGROUND: Monitoring the adaptive immune responses during the natural course of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection provides useful information for the development of vaccination strategies against this virus and its emerging variants. We thus profiled the serum anti-SARS-CoV-2 antibody (Ab) levels and specific memory B and T cell responses in convalescent coronavirus disease 2019 (COVID-19) patients. METHODS: A total of 119 samples from 88 convalescent donors who experienced mild to critical disease were tested for the presence of elevated anti-spike and anti-receptor binding domain Ab levels over a period of 8 months. In addition, the levels of SARS-CoV-2 neutralizing Abs and specific memory B and T cell responses were tested in a subset of samples. FINDINGS: Anti-SARS-CoV-2 Abs were present in 85% of the samples collected within 4 weeks after the onset of symptoms in COVID-19 patients. Levels of specific immunoglobulin M (IgM)/IgA Abs declined after 1 month, while levels of specific IgG Abs and plasma neutralizing activities remained relatively stable up to 6 months after diagnosis. Anti-SARS-CoV-2 IgG Abs were still present, although at a significantly lower level, in 80% of the samples collected at 6-8 months after symptom onset. SARS-CoV-2-specific memory B and T cell responses developed with time and were persistent in all of the patients followed up for 6-8 months. CONCLUSIONS: Our data suggest that protective adaptive immunity following natural infection of SARS-CoV-2 may persist for at least 6-8 months, regardless of disease severity. Development of medium- or long-term protective immunity through vaccination may thus be possible. FUNDING: This project was supported by the European Union's Horizon 2020 research and innovation programme (ATAC, no. 101003650), the Italian Ministry of Health (Ricerca Finalizzata grant no. GR-2013-02358399), the Center for Innovative Medicine, and the Swedish Research Council. J.A. was supported by the SciLifeLab/KAW national COVID-19 research program project grant 2020.

Vitiligo susceptibility and catalase gene polymorphisms in Sicilian population.

BACKGROUND: Catalase gene (CAT) polymorphisms were analyzed as responsible for the deficiency of catalase enzyme activity and concomitant accumulation of excessive hydrogen peroxide in vitiligo patients. Catalase is a well-known oxidative stress regulator that could play an important role in the pathogenesis of vitiligo. This study was conducted to evaluate three CAT gene polymorphisms (-89A/T, 389C/T, 419C/T) and their association with vitiligo susceptibility in Sicilian population. METHODS: Sixty out of 73 Sicilian patients with vitiligo were enrolled and submitted to CAT gene analysis. RESULTS: Contrary to the Northern part of Europe but likewise to the Mediterranean area, the frequency of the CAT genotypes in Sicily is equally distributed. Out of all CAT genotypes, only CAT-89 T/T frequency was found to be significantly higher amongst vitiligo patients than controls. CONCLUSIONS: Despite the involvement of the CAT enzyme in the pathogenesis of vitiligo, the biological significance of CAT gene polymorphisms is still controversial. With the only exception for CAT variant -89A/T, the other studied CAT gene polymorphisms (389C/T and 419C/T) might not to be associated with vitiligo in Sicilian population.

A clinical case of sepsis due to Staphylococcus pettenkoferi.

Coagulase-negative staphylococci are part of the human skin flora but are frequently responsible for bloodstream infection, especially in the presence of intravascular devices or immunosuppressive conditions. Antibiotic resistance in such bacteria is common, with more than 80% of isolates resistant to methicillin. Among this genus Staphylococcus pettenkoferi is a recently identified organism, reported to be responsible for a growing number of infections. Here we describe a case of sepsis due to methicillin-resistant S. pettenkoferi.

Febrile rhabdomyolysis of unknown origin in refugees coming from West Africa through the Mediterranean.

OBJECTIVES: Cases of undiagnosed severe febrile rhabdomyolysis in refugees coming from West Africa, mainly from Nigeria, has been observed since May 2014. The aim of this study was to describe this phenomenon. METHODS: This was a multicentre retrospective observational study of cases of febrile rhabdomyolysis reported from May 2014 to December 2016 in 12 Italian centres. RESULTS: A total of 48 cases were observed, mainly in young males. The mean time interval between the day of departure from Libya and symptom onset was 26.2 days. An average 8.3 further days elapsed before medical care was sought. All patients were hospitalized with fever and very intense muscle aches. Creatine phosphokinase, aspartate aminotransferase, and lactate dehydrogenase values were abnormal in all cases. The rhabdomyolysis was ascribed to an infective agent in 16 (33.3%) cases. In the remaining cases, the aetiology was undefined. Four out of seven patients tested had sickle cell trait. No alcohol abuse or drug intake was reported, apart from a single reported case of khat ingestion. CONCLUSIONS: The long incubation period does not support a mechanical cause of rhabdomyolysis. Furthermore, viral infections such as those caused by coxsackievirus are rarely associated with such a severe clinical presentation. It is hypothesized that other predisposing conditions like genetic factors, unknown infections, or unreported non-conventional remedies may be involved. Targeted surveillance of rhabdomyolysis cases is warranted.

Monitoring renal function during combination therapy with telaprevir in HIV/HCV co-infected patients with advanced/fibrosis cirrhosis.

INTRODUCTION: Telaprevir (TVR) plus peg interferon (PEG-IFN) and ribavirin (RBV) substantially increase treatment efficacy for genotype 1 chronic hepatitis C virus (HCV) infection but data about its safety in HIV patients with cirrhosis are lacking. Our purpose was to evaluate estimated Glomerular Filtration Rates (eGFR) variations during combination therapy in a difficult-to-treat HIV/HCV population with advanced fibrosis/cirrhosis through three different scores commonly used in clinical practice: CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration), Modification of Diet in Renal Disease (MDRD) scores (that consider gender, ethnicity, age, serum creatinine level [SCL]) and MDRD 6 variable (that considers the previous parameters plus blood urea nitrogen and serum albumin). Second objective was to identify any association between creatinin clearance and haemoglobin (Hb) variation during combination therapy. MATERIALS AND METHODS: We conducted an observational retrospective study including 18 HIV/HCV patients attending our clinic who started combination therapy for HCV, including in the analysis the first 16 weeks of therapy. Treatment included TVR 1125 mg BID (twice a day) for 12 weeks, PEG-IFN α-2a 180 mcg QW and RBV daily dose according to body weight (800 mg in individuals <60 kg; 1000 mg in individuals 60-75 kg; 1200 mg in individuals >75 kg). Advanced fibrosis was defined as Metavir score=F3 or Ishak 3-4 and cirrhosis was defined as Metavir score F4 or Ishak 5-6 assessed by liver biopsy or transient elastography respectively. P per trend were assessed to evaluate any change of SCL and eGFR (according to different formulas). Multilevel linear regression analysis was performed to estimate factors associated with reduction of Hb. RESULTS: Baseline characteristics and HCV virological responses were collected. Figure 1 shows median SCL and eGFR trends across follow-up period: SCR p-per-trend was 0.28, for CKD-EPI was 0.50, for MDRD was 0.48, for MDRD-6 variables was 0.27. Hb trend was also assessed and a significant decrease of Hb across follow-up (p per trend <0.001) was noted. Multilevel regression analysis found an association between SCL and Hb change, after adjustment for age, sex, basal BMI, tenofovir exposure and proteinuria (assessed at baseline) as shown in Table 1. CONCLUSIONS: In our experience, combination therapy with TPV in cirrhotic HIV-HCV patients did not significantly affect renal function. As expected, Hb levels decreased during treatment and it is likely related to RBV exposure. In multivariable analysis, reduction of Hb appeared to be related to SCL higher levels, thus suggesting even a mild decrease of renal function.

T cell activation but not polyfunctionality after primary HIV infection predicts control of viral load and length of the time without therapy.

OBJECTIVE: Immune changes occurring after primary HIV infection (PHI) have a pivotal relevance. Our objective was to characterize the polyfunctionality of immune response triggered by PHI, and to characterize immune activation and regulatory T cells, correlating such features to disease progression. PATIENTS AND METHODS: We followed 11 patients experiencing PHI for 4 years. By polychromatic flow cytometry, we studied every month, for the first 6 months, T lymphocyte polyfunctionality after cell stimulation with peptides derived from HIV-1 gag and nef. Tregs were identified by flow cytometry, and T cell activation studied by CD38 and HLA-DR expression. RESULTS: An increase of anti-gag and anti-nef CD8+ specific T cells was observed 3 months after PHI; however, truly polyfunctional T cells, also able to produce IL-2, were never found. No gross changes in Tregs were present. T lymphocyte activation was maximal 1 and 2 months after PHI, and significantly decreased in the following period. The level of activation two months after PHI was strictly correlated to the plasma viral load 1 year after infection, and significantly influenced the length of period without therapy. Indeed, 80% of patients with less than the median value of activated CD8+ (15.5%) or CD4+ (0.9%) T cells remained free of therapy for >46 months, while all patients over the median value had to start treatment within 26 months. CONCLUSIONS: T cell activation after PHI, more than T cell polyfunctionality or Tregs, is a predictive marker for the control of viral load and for the time required to start treatment.