Coordinated innate and T-cell immune responses in mild COVID-19 patients from household contacts of COVID-19 cases during the first pandemic wave.

Objective: To better define the immunopathogenesis of COVID-19, the present study aims to characterize the early immune responses to SARS-CoV-2 infection in household contacts of COVID-19 cases. In particular, innate, T- and B-cell specific responses were evaluated over time. Methods: Household contacts of COVID-19 cases screened for SARS-CoV-2 infection by nasopharyngeal swab for surveillance purposes were enrolled (T0, n=42). Of these, 28 subjects returned for a follow-up test (T1). The innate response was assessed by detecting a panel of soluble factors by multiplex-technology in plasma samples. Cell-mediated response was evaluated by measuring interferon (IFN)-γ levels by ELISA in plasma harvested from whole-blood stimulated with SARS-CoV-2 peptide pools, including spike (S), nucleocapsid (N) and membrane (M) proteins. The serological response was assessed by quantifying anti-Receptor-Binding-Domain (RBD), anti-Nucleocapsid (N), whole virus indirect immunofluorescence, and neutralizing antibodies. Results: At T0, higher levels of plasmatic IFN-α, IL-1ra, MCP-1 and IP-10, and lower levels of IL-1ß, IL-9, MIP-1ß and RANTES were observed in subjects with positive swab compared to individuals with a negative one (p<0.05). Plasmatic IFN-α was the only cytokine detectable in subjects with positive SARS-CoV-2 swabs with high accuracy for swab score positivity (0.93, p<0.0001). Among subjects with positive swabs, significant negative correlations were found among the RT-PCR cycle threshold values reported for genes S and N and IFN-α or IP-10 levels. At T0, the IFN-γ T-cell specific response was detected in 50% (5/10) of subjects with positive swab, while anti-RBD/anti-N antibodies showed a positivity rate of 10% (1/10). At T1, the IFN-γ T-cell specific response was detected in most of the confirmed-infection subjects (77.8%, 7/9), whereas the serological response was still observed in a minority of them (44.4%, 4/9). Overall, the swab test showed a moderate concordance with the T-cell response (78.6%, k=0.467), and a scarce concordance with the serological one (72.9%, k=0.194). Conclusions: Plasmatic IFN-α and the IFN-γ T-cell specific response appear early even in the absence of seroconversion, and show a greater positivity rate than the serological response in household contacts with positive swab.

Hospitalized patients with diarrhea: Rate of Clostridioides difficile infection underdiagnosis and drivers of clinical suspicion.

OBJECTIVES: Clostridioides difficile infection (CDI) represents a challenging issue, with an evolving epidemiology. Main objectives of our study were: to assess the frequency of diarrhea of overall etiology, including CDI, as a cause of hospital admission or occurring during hospital stay;- to determine the rate of underdiagnosis of community-acquired (CA-), health care associated (HCA)- and hospital onset (HO-) CDI, and explore factors associated with its clinical suspicion by physicians. METHODS: A prospective cohort study included all hospitalized patients with diarrhea at two acute-care hospitals. C. difficile (CD) tests were performed on every stool samples, irrespective of the treating physician request. Factors associated with the likelihood of CD test request by physicians were assessed. RESULTS: We enrolled 871 (6%) patients with diarrhea. CD test performed on all diarrheic stool samples was positive in 228 cases (26%); 37, 106, 85 cases of CA- (14%), HCA- (42%) and HO- diarrhea (24%), respectively. Treating physicians did not request CD test in 207 (24%) diarrhea cases. The rate of CDI underdiagnosis was 11% (24/228); it was higher in CA-CDI (27%, 10/37). Logistic regression analysis identified age >65 years (RR 1.1; 95 CI 1.06-1.2) and hospitalizations in the previous 3 months (RR 1.2; 95% CI 1.1-1.3) as independent factors associated with the likelihood of requesting the CD test by the physician. These risk factors differed by epidemiological classification of diarrhea and by hospital. CONCLUSIONS: Our study confirmed the relevance of CDI underdiagnosis and provided new insights in the factors underlying the lack of CDI clinical suspicion.

Molecular analysis of clinical isolates of ceftazidime-avibactam-resistant Klebsiella pneumoniae.

OBJECTIVES: To analyse the strains collected during a 1-year survey of ceftazidime-avibactam-resistant KPC-producing Klebsiella pneumoniae, in order to investigate the molecular mechanisms potentially responsible for their resistant phenotype. METHODS: Clinical KPC-producing K. pneumoniae isolates were collected from 31 patients in six different hospitals in Rome. For eight of the patients, an additional strain grown before the start of treatment was also available, bringing the total of isolates studied to 39. Antimicrobial susceptibility was determined by automated system, broth microdiluition and E-test as appropriate. In silico analysis of acquired resistance genes was achieved by whole-genome sequencing, while multilocus sequence typing and core genome multilocus sequence typing were employed for molecular typing. Mutations associated with ceftazidime-avibactam resistance were identified by Sanger sequencing of the blaKPC gene. Possible mutations in OmpK35 and OmpK36 outer membrane proteins were also investigated. RESULTS: Molecular analyses highlighted the circulation of the ST512, 101 and 307 high-risk clones; 26 of the 31 patients carried a mutated KPC variant, five had a wild-type KPC-3. Among the KPC variants detected, 11 were different mutations within the blaKPC-3 gene, four of which were novel mutational changes. CONCLUSIONS: Different mutations including single amino-acid substitutions, insertions or deletions within the blaKPC gene were found in 26/31 ceftazidime-avibactam-resistant KPC-producing K. pneumoniae strains belonging to high-risk clones circulating in Italy. Of note, in 14/31 cases the isolates displayed resistance to both ceftazidime-avibactam and carbapenems, raising concerns for the possible selection of a multidrug-resistant phenotype.

Trends in the Incidence and Antibiotic Resistance of Enterococcal Bloodstream Isolates: A 7-Year Retrospective Multicenter Epidemiological Study in Italy.

The spread of resistance to vancomycin and other last-resort drugs in Enterococcus spp. remains of concern. In Italy, surveillance data for enterococcal bloodstream isolates in humans are scant. The aim of our study was to assess the incidence trends of bacteremias due to Enterococcus species and their prevalence trends of antimicrobial resistance. We retrospectively included all consecutive not-duplicate Enterococcus species isolated from blood cultures, in patients from 11 Italian hospitals (2011-2017). Incidence was defined as the number of isolates per 10,000 patient-days, while resistance prevalence was defined as the number of resistant strains divided by the number of tested strains. We included 4,858 isolates (59%, 36%, and 5% due to Enterococcus faecalis, E. faecium, and other Enterococcus spp., respectively). Over the study period, the incidence of bacteremias due to E. faecalis (incidence rate ratio [IRR]: 1.02, 95% confidence interval [CI]: 1.00-1.04, p = 0.008) and E. faecium increased (IRR: 1.03, 95% CI: 1.01-1.05, p < 0.001) alongside with the whole enterococcal bacteremias trend (IRR: 1.02, 95% CIs: 1.01-1.04, p = 0.002). A progressive increase in vancomycin-resistant E. faecium (VREfm) bacteremias was observed. Resistance to tigecycline and linezolid was rarely reported. The incidence of enterococcal bloodstream isolates is increasing in Italy, together with the prevalence of VREfm. Resistance to linezolid, a cornerstone drug used in the treatment of VRE bloodstream infection, remains negligible.

A proposal for a comprehensive approach to infections across the surgical pathway.

Despite evidence supporting the effectiveness of best practices in infection prevention and management, many healthcare workers fail to implement them and evidence-based practices tend to be underused in routine practice. Prevention and management of infections across the surgical pathway should always focus on collaboration among all healthcare workers sharing knowledge of best practices. To clarify key issues in the prevention and management of infections across the surgical pathway, a multidisciplinary task force of experts convened in Ancona, Italy, on May 31, 2019, for a national meeting. This document represents the executive summary of the final statements approved by the expert panel.

Use of WHONET-SaTScan system for simulated real-time detection of antimicrobial resistance clusters in a hospital in Italy, 2012 to 2014.

Resistant pathogens infections cause in healthcare settings, higher patient mortality, longer hospitalisation times and higher costs for treatments. Strengthening and coordinating local, national and international surveillance systems is the cornerstone for the control of antimicrobial resistance (AMR). In this study, the WHONET-SaTScan software was applied in a hospital in Italy to identify potential outbreaks of AMR. Data from San Filippo Neri Hospital in Rome between 2012 and 2014 were extracted from the national surveillance system for antimicrobial resistance (AR-ISS) and analysed using the simulated prospective analysis for real-time cluster detection included in the WHONET-SaTScan software. Results were compared with the hospital infection prevention and control system. The WHONET-SaTScan identified 71 statistically significant clusters, some involving pathogens carrying multiple resistance phenotypes. Of these 71, three were also detected by the hospital system, while a further 15, detected by WHONET-SaTScan only, were considered of relevant importance and worth further investigation by the hospital infection control team. In this study, the WHONET-SaTScan system was applied for the first time to the surveillance of AMR in Italy as a tool to strengthen this surveillance to allow more timely intervention strategies both at local and national level, using data regularly collected by the Italian national surveillance system.

In Vitro Activity of Tea Tree Oil Vaginal Suppositories against Candida spp. and Probiotic Vaginal Microbiota.

The aim of this work is to evaluate the in vitro microbicidal activity of vaginal suppositories (VS) containing tea tree oil (TTO-VS) towards Candida spp. and vaginal probiotics. A total of 20 Candida spp. strains, taken from patients with vaginitis and from an established type collection, including reference strains, were analysed by using the CLSI microdilution method. To study the action of VS towards the beneficial vaginal microbiota, the sensitivity of Bifidobacterium animalis subsp. lactis (DSM 10140) and Lactobacillus spp. (Lactobacillus casei R-215 and Lactobacillus acidophilus R-52) was tested. Both TTO-VS and TTO showed fungicidal activity against all strains of Candida spp. whereas placebo-VS or the Aloe gel used as controls were ineffective. The study of fractional fungicidal concentrations (FFC) showed synergistic interaction with the association between Amphotericin B and TTO (0.25 to 0.08 µg/ml, respectively) against Candida albicans. Instead, the probiotics were only affected by TTO concentration ≥ 4% v/v, while, at concentrations < 2% v/v, they remained viable. TTO-VS exhibits, in vitro, a selective fungicidal action, slightly affecting only the Bifidobacteriun animalis strain growth belonging to the vaginal microbiota. In vivo studies are needed to confirm the efficacy to prevent acute or recurrent vaginal candidiasis.

Microbiologic characteristics and predictors of mortality in bloodstream infections in intensive care unit patients: A 1-year, large, prospective surveillance study in 5 Italian hospitals.

BACKGROUND: Bloodstream infections (BSIs) from multidrug-resistant (MDR) bacteria cause morbidity and mortality in intensive care unit (ICU) patients worldwide. This study investigated the incidence of BSIs in 5 adult general ICUs in Rome, Italy, and evaluated the mortality rate and risk factors associated with these infections. METHODS: Over a 12-month period, 1,318 patients were enrolled. Demographic characteristics, Simplified Acute Physiology Score II (SAPS II), comorbidities, and BSI isolate data were collected. After stratification for the outcome, statistical analysis was performed to assess the impact of patient risk factors on in-hospital mortality. RESULTS: There were 324 BSIs in 175 patients recorded, with an in-hospital mortality rate of 46%. Univariate analysis revealed that SAPS II, cardiac comorbidity, and Klebsiella pneumoniae BSI were significantly associated with a higher risk of death. Having a K pneumoniae BSI and cardiac illness at admission were both confirmed to be associated with death by multivariate analysis (P = .0162 and P = .0158, respectively). Most of the K pneumoniae isolates showed high resistance rates to carbapenems. CONCLUSION: BSIs caused by K pneumoniae and cardiovascular comorbidity in ICU patients are associated with a higher risk of death. Thorough surveillance for MDR pathogens and stratification of the patients' risk on admission into the ICU are key to improving the outcomes of these infections.

Elevated antinuclear antibodies and altered anti-Epstein-Barr virus immune responses.

It has been shown that Epstein-Barr virus (EBV) is able to alter the immune response towards self-antigens and may enhance risk of autoimmune diseases such as systemic lupus erythematosus (SLE) in genetically predisposed individuals. In this study, we evaluated the specific antibody immune response against EBV in patients with anti-nuclear autoantibodies (ANA) in comparison with ANA-negative healthy controls. For this purpose, 92 patients with an high anti-ANA reactivity with or without concomitant extractable nuclear antigen (ENA) or double stranded DNA (dsDNA) positivity were selected and compared with 146 healthy donors. We found that anti-EBV-VCA and EA IgG concentrations were significantly higher in ANA-positive patients in comparison to the controls (VCA P<0.0001 and EA P<0,03) as well as in those ANA-positive patients that showed a concomitant ENA positivity (P=0.0002). Interestingly, elevated anti-EBNA-1 IgG was found in a group of patients who had anti SSA/Ro antibodies. Anti-VCA IgM Abs were more frequently found in those patients with a very high titer of ANA (P=0.06); moreover detection of anti-VCA IgM/IgG in absence of anti-EBNA-1 IgG was more frequent in the patient than in the control group. Both these conditions correlate with a recent EBV infection or reactivation. The data suggest that EBV, particularly during acute infection or in its reactivation phase, could be involved in the ANA and ENA autoantibody formation.

Pandemic influenza A/H1N1pdm in Italy: age, risk and population susceptibility.

BACKGROUND: A common pattern emerging from several studies evaluating the impact of the 2009 A/H1N1 pandemic influenza (A/H1N1pdm) conducted in countries worldwide is the low attack rate observed in elderly compared to that observed in children and young adults. The biological or social mechanisms responsible for the observed age-specific risk of infection are still to be deeply investigated. METHODS: The level of immunity against the A/H1N1pdm in pre and post pandemic sera was determined using left over sera taken for diagnostic purposes or routine ascertainment obtained from clinical laboratories. The antibody titres were measured by the haemagglutination inhibition (HI) assay. To investigate whether certain age groups had higher risk of infection the presence of protective antibody (≥1∶40), was calculated using exact binomial 95% CI on both pre- and post- pandemic serological data in the age groups considered. To estimate age-specific susceptibility to infection we used an age-structured SEIR model. RESULTS: By comparing pre- and post-pandemic serological data in Italy we found age- specific attack rates similar to those observed in other countries. Cumulative attack rate at the end of the first A/H1N1pdm season in Italy was estimated to be 16.3% (95% CI 9.4%-23.1%). Modeling results allow ruling out the hypothesis that only age-specific characteristics of the contact network and levels of pre-pandemic immunity are responsible for the observed age-specific risk of infection. This means that age-specific susceptibility to infection, suspected to play an important role in the pandemic, was not only determined by pre-pandemic levels of H1N1pdm antibody measured by HI. CONCLUSIONS: Our results claim for new studies to better identify the biological mechanisms, which might have determined the observed pattern of susceptibility with age. Moreover, our results highlight the need to obtain early estimates of differential susceptibility with age in any future pandemics to obtain more reliable real time estimates of critical epidemiological parameters.

Clostridium difficile infection in Italian urban hospitals: data from 2006 through 2011.

BACKGROUND: In developed countries, Clostridium difficile infection (CDI) represents an emerging threat in terms of morbidity and mortality rates. In our country limited CDI epidemiological data can be found. METHODS: Stool samples tested for C. difficile toxins from January 2006 to December 2011 in 5 large hospitals in Rome, Italy, were considered in the analysis. Repeated samples taken ≤ 2 months after a positive result were excluded. RESULTS: A total of 402 CDI episodes were identified. The incidence of CDI episodes progressively increased from 0.3 in 2006 to 2.3 per 10,000 patient-days in 2011. CDI episodes mostly occurred in patients > 60 years of age (77%). The >80 year-old age class reported the highest percentage of CDI episodes on tested samples (16%). Eighty percent (80%) of CDI episodes occurred in medical wards followed by surgery (10.2%) and intensive care units (9.8%). CONCLUSIONS: A significant increasing incidence of CDI episodes over the study period was observed during the years (p<.001), particularly in the older age groups. Medical wards experienced the highest number of CDI episodes as compared to intensive care and surgical wards. The increasing rate of CDI episodes over the last six years in our country, is alarming; urgent improvements in the surveillance systems and control programs are advisable.

Daptomycin plus trimethoprim/sulfamethoxazole combination therapy in post-neurosurgical meningitis caused by linezolid-resistant Staphylococcus epidermidis.

Post-neurosurgical infection is a serious complication that occurs in approx. 4% of all patients undergoing neurosurgical procedures and is associated with high morbidity and mortality rates and prolonged length of intensive care unit (ICU) stay. Coagulase-negative staphylococci (CoNS), especially methicillin-resistant Staphylococcus epidermidis (MRSE), are the most frequent pathogens involved in CNS post-neurosurgical meningitis. Treatment is challenging especially in patients with meningitis due to multidrug- resistant (MDR) CONS. Herein, we report a unique case of post-neurosurgical meningitis due to MRSE resistant to linezolid (a molecular analysis revealed the presence of the mutation G2576T on domain V of the 23S rRNA gene) and with reduced susceptibility to glycopeptides, successfully treated with a combination of daptomycin at 10 mg/kg daily plus trimethoprim/sulfamethoxazole (TMP/SMX). This antibiotic combination showed an indifferent interaction in in vitro studies. Daptomycin serum and cerebrospinal fluid (CSF) concentrations, determined through blood and CSF samples drawn just prior to and 4 h after the third dose, were 18.9-0.78 and 51.65-3.1 mg/L, respectively. These values allowed us to approximate a 5-6% penetration rate of the drug through an inflamed blood-brain barrier. In conclusion, although further studies are needed, combination of high-dose daptomycin plus TMP/SMX is a reasonable option for treatment of meningitis caused by multidrug-resistant S. epidermidis.

Diagnostic difficulties of Lactobacillus casei bacteraemia in immunocompetent patients: a case report.

INTRODUCTION: Lactobacilli are currently proposed as probiotic agents in several dietary products. In blood cultures, they are usually considered as contaminants, but in recent years they have been recognized as causal infectious agents of endocarditis, urinary tract infections, meningitis, intra-abdominal infections and bacteraemia. CASE PRESENTATION: We report a case of Lactobacillus casei bacteraemia in a 66-year-old immunocompetent man with a history of fever of unknown origin. Leuconostoc bacteraemia was demonstrated by blood culture, but a later polymerase chain reaction analysis with sequencing of 16S ribosomal RNA identified Lactobacillus casei and a successful antibiotic therapy was performed. CONCLUSION: Bacteraemia caused by probiotic organisms is rare but underestimated, since they are normally regarded as contaminants and their role as primary invaders is not always easily established. Although the consumption of probiotic products cannot be considered a risk factor in the development of diseases caused by usually non-pathogenic bacteria, specific individual clinical histories should be taken into account. This report should alert both clinicians and microbiologists to the possibility of unusual pathogens causing serious illnesses and to the use of 16S ribosomal RNA sequencing for molecular identification as a powerful tool in confirming the diagnosis of infrequent pathogens.

Decrease of methicillin resistant Staphylococcus aureus prevalence after introduction of a surgical antibiotic prophylaxis protocol in an Italian hospital.

The use of antimicrobials is an important factor contributing to the emergence of antibiotic resistance. The goal of our study was to evaluate the impact of the introduction of an antibiotic surgical prophylaxis protocol on the prevalence of methicillin resistant Staphylococcus aureus (MRSA) infections in a tertiary care hospital. The protocol of surgical antibiotic prophylaxis was designed by a multidisciplinary team and was implemented in December 2001. Between January 2002 and December 2002, pharmacy, laboratory and active surveillance-records were prospectively reviewed to calculate prevalence rates of defined daily doses (DDD), microorganism isolation and health-care related infections. A progressive decrease from 1.58 to 0.56 of MRSA isolations per 1000 patient-days and from 76.4% to 29.4% MRSA prevalence rate was reported (p<0.001). Monthly prevalence rates of MRSA showed a significant linear correlation with the reduction of the DDD of the 3rd generation cephalosporins (r=0.90; p<0.001). MRSA surgical site and blood stream infections decreased from 78% to 38% and from 89% to 38%, respectively (p=0.017 and p=0.026). In our experience, the reduction of 3rd generation cephalosporin use was an effective strategy to reduce the MRSA infection rate and was associated with the reduction of the overall expenditure for antibiotics in the hospital.