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Objectives: HIV-1 infection continues to be a significant public health concern, notwithstanding the expanded utilization of antiretroviral treatment (ART), due to the emergence of drug resistance. The prevalence of transmitted drug resistance remains uncertain, particularly concerning integrase inhibitors. This study aimed to assess the extent of HIV resistance in both ART-naïve and experienced individuals living with HIV (PLHIV) at the University Hospital in Palermo, Italy., Methods: Genotyping and mutation analysis were performed on ART naïve and experienced PLHIV admitted from June 2021 to October 2023 by the NGS method. Mutations were detected by testing different NGS frequency cut-offs: ≥5 %, ≥10 %, and ≥20 %. Demographic, clinical, virological, and immunological data were retrospectively collected., Results: Of the PLHIV, 85 (70 %) were ART-naïve, while 36 (30 %) were ART-experienced with virological failure. The main HIV-1 subtype was B (54 %), which was significantly associated with Italy-born (P < 0.001) and experienced PLHIV (P = 0.024). In the remaining cases, A1 (6 %), C (3 %), F1 (7 %), G (2 %), and Circulating Recombinant Forms (28 %) were reported. At least one mutation for a drug class was detected in 39.7 %, 45.4 %, and 53.7 % of cases at HIV-1 NGS thresholds of 20 %, 10 %, and 5 %, respectively. Drug resistance was found in 18.2 %, 25.6 %, and 33.0 %, by NGS cut-off of 20 %, 10 %, and 5 % respectively. The lowering of NGS cut-offs mainly increased the rates of integrase strand transfer inhibitor resistance. For overall resistance, no difference was observed between B and non-B subtypes for any NGS cut-offs., Competing Interests: Declaration of competing interest None declared., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Bloodstream infections (BSIs) and sepsis are a major cause of morbidity and mortality. Appropriate early antibiotic therapy is crucial for improving the survival of patients with sepsis and septic shock. T2 magnetic resonance (T2MR ® ) technology may enable fast and sensitive detection of ESKAPEc pathogens directly from whole-blood samples. We aimed to evaluate concordance between the T2Bacteria ® Panel and standard blood culture and its impact on antibiotic therapy decisions. We conducted a single-centre retrospective study on patients with sepsis-induced hypotension or septic shock admitted to general, post-operative/neurosurgical, and cardiothoracic Intensive Care Units who were tested with the T2Bacteria ® Panel from January 2021 to December 2022. Eighty-five consecutively admitted patients were included, for a total of 85 paired tests. A total of 48 ESKAPEc pathogens were identified by the T2Bacteria ® Panel. The concordance rate between the T2Bacteria ® Panel and blood cultures was 81% (69/85), with 20 concordant-positive and 49 concordant-negative cases. For the 25 microorganisms grown from accompanying blood cultures, blood pathogen coverage by the T2Bacteria ® Panel was 88%. In this cohort of severely ill septic patients, the T2Bacteria ® Panel was highly concordant and was able to detect more ESKAPEc pathogens, with a significantly shorter turn-around time compared to conventional blood cultures. The T2Bacteria ® Panel also significantly impacted decisions on antibiotic therapy.

Background: Next-generation sequencing (NGS) is gradually replacing Sanger sequencing for HIV genotypic drug resistance testing (GRT). This work evaluated the concordance among different NGS-GRT interpretation tools in a real-life setting., Methods: Routine NGS-GRT data were generated from viral RNA at 11 Italian laboratories with the AD4SEQ HIV-1 Solution v2 commercial kit. NGS results were interpreted by the SmartVir system provided by the kit and by two online tools (HyDRA Web and Stanford HIVdb). NGS-GRT was considered valid when the coverage was >100 reads (100×) at each PR/RT/IN resistance-associated position listed in the HIVdb 9.5.1 algorithm., Results: Among 629 NGS-GRT, 75.2%, 74.2%, and 70.9% were valid according to SmartVir, HyDRA Web, and HIVdb. Considering at least two interpretation tools, 463 (73.6%) NGS-GRT had a valid coverage for resistance analyses. The proportion of valid samples was affected by viremia <10,000-1000 copies/mL and non-B subtypes. Mutations at an NGS frequency >10% showed fair concordance among different interpretation tools., Conclusion: This Italian survey on NGS resistance testing suggests that viremia levels and HIV subtype affect NGS-GRT coverage. Within the current routine method for NGS-GRT, only mutations with frequency >10% seem reliably detected across different interpretation tools.

Although the anti-COVID-19 vaccination has proved to be an effective preventive tool, "breakthrough infections" have been documented in patients with complete primary vaccination courses. Most of the SARS-CoV-2 neutralizing antibodies produced after SARS-CoV-2 infection target the spike protein receptor-binding domain which has an important role in facilitating viral entry and the infection of the host cells. SARS-CoV-2 has demonstrated the ability to evolve by accumulating mutations in the spike protein to escape the humoral response of a host. The aim of this study was to compare the titers of neutralizing antibodies (NtAbs) against the variants of SARS-CoV-2 by analyzing the sera of recovered and vaccinated healthcare workers (HCWs). A total of 293 HCWs were enrolled and divided into three cohorts as follows: 91 who had recovered from SARS-CoV-2 infection (nVP); 102 that were vaccinated and became positive after the primary cycle (VP); and 100 that were vaccinated with complete primary cycles and concluded the follow-up period without becoming positive (VN). Higher neutralization titers were observed in the vaccinated subjects' arms compared to the nVP subjects' arms. Differences in neutralization titers between arms for single variants were statistically highly significant ( p < 0.001), except for the differences between titers against the Alpha variant in the nVP and in VP groups, which were also statistically significant ( p < 0.05). Within the nVP group, the number of subjects with an absence of neutralizing antibodies was high. The presence of higher titers in patients with a complete primary cycle compared to patients who had recovered from infection suggested the better efficacy of artificial immunization compared to natural immunization, and this further encourages the promotion of vaccination even in subjects with previous infections., Competing Interests: The authors declare no conflict of interest.

CD28-driven "signal 2" is critical for naïve CD8 + T cell responses to dendritic cell (DC)-presented weak antigens, including non-mutated tumor-associated antigens (TAAs). However, it is unclear how DC-primed cytotoxic T lymphocytes (CTLs) respond to the same TAAs presented by cancer cells which lack CD28 ligands. Here, we show that NK receptors (NKRs) DNAM-1 and NKG2D replace CD28 during CTL re-activation by cancer cells presenting low levels of MHC I/TAA complexes, leading to enhanced proximal TCR signaling, immune synapse formation, CTL polyfunctionality, release of cytolytic granules and antigen-specific cancer cell killing. Double-transduction of T cells with recombinant TCR and NKR constructs or upregulation of NKR-ligand expression on cancer cells by chemotherapy enabled effective recognition and killing of poorly immunogenic tumor cells by CTLs. Operational synergy between TCR and NKRs in CTL recognition explains the ability of cancer-expressed self-antigens to serve as tumor rejection antigens, helping to develop more effective therapies., Competing Interests: Conflict of interest: The authors have declared that no conflict of interest exists.

Aims/hypothesis: Islet autoantibodies (AAbs) are detected in >90% of individuals with clinically suspected type 1 diabetes at disease onset. A single AAb, sometimes at low titre, is often detected in some individuals, making their diagnosis uncertain. Type 1 diabetes genetic risk scores (GRS) are a useful tool for discriminating polygenic autoimmune type 1 diabetes from other types of diabetes, particularly the monogenic forms, but testing is not routinely performed in the clinic. Here, we used a type 1 diabetes GRS to screen for monogenic diabetes in individuals with weak evidence of autoimmunity, i.e. with a single AAb at disease onset., Methods: In a pilot study, we genetically screened 142 individuals with suspected type 1 diabetes, 42 of whom were AAb-negative, 27 of whom had a single AAb (single AAb-positive) and 73 of whom had multiple AAbs (multiple AAb-positive) at disease onset. Next-generation sequencing (NGS) was performed in 41 AAb-negative participants, 26 single AAb-positive participants and 60 multiple AAb-positive participants using an analysis pipeline of more than 200 diabetes-associated genes., Results: The type 1 diabetes GRS was significantly lower in AAb-negative individuals than in those with a single and multiple AAbs. Pathogenetic class 4/5 variants in MODY or monogenic diabetes genes were identified in 15/41 (36.6%) AAb-negative individuals, while class 3 variants of unknown significance were identified in 17/41 (41.5%). Residual C-peptide levels at diagnosis were higher in individuals with mutations compared to those without pathogenetic variants. Class 3 variants of unknown significance were found in 11/26 (42.3%) single AAb-positive individuals, and pathogenetic class 4/5 variants were present in 2/26 (7.7%) single AAb-positive individuals. No pathogenetic class 4/5 variants were identified in multiple AAb-positive individuals, but class 3 variants of unknown significance were identified in 19/60 (31.7%) patients. Several patients across the three groups had more than one class 3 variant., Conclusions/interpretation: These findings provide insights into the genetic makeup of patients who show weak evidence of autoimmunity at disease onset. Absence of islet AAbs or the presence of a single AAb together with a low type 1 diabetes GRS may be indicative of a monogenic form of diabetes, and use of NGS may improve the accuracy of diagnosis., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Aims: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection has had a serious worldwide impact on human health. On December 2020, an immunization campaign with a COVID-19 mRNA vaccine (Comirnaty-BNT162b2 Pfizer-BioNTech) was started in Italy, first targeting healthcare workers (HCWs). This study aims to investigate the antibodies that are response against SARS-CoV-2 vaccine., Methods and Results: The kinetics and the persistence of both anti-S1/S2 IgGs and neutralizing antibodies (Nt-Abs) were investigated in 76 HCWs through a 4-month follow-up with multiple testing points starting at the first dose. Temporal analysis of SARS-CoV-2 Abs titre kinetics showed three different stages, with an initial slow growth in the anti-S1/S2 IgGs and Nt-Abs titres, corresponding to the first 4 weeks after the first dose of vaccine, followed by a second stage with peaks in titres, around 35 days after the first dose, and by a third stage (38 to 90-120 days after the first dose) showing a steady decrease in anti-S1/S2 IgGs while Nt-Abs are maintained at stable levels. Moreover, the levels of specific Nt-Abs to SARS-CoV-2 Spike protein are correlated to the anti-S1/S2 IgG titre (R-squared = 0.47; p < 0.001)., Conclusions: The levels of specific Nt-Abs to SARS-CoV-2 Spike protein are correlated to the anti-S1/S2 IgG titre, although Nt-Abs could maintain a more stable titre over the time despite declining IgG Abs titre., Significance and Impact: This study highlights the kinetics and the persistence of Nt-Abs in HCWs vaccinated with Comirnaty (BNT162b2) Pfizer-BioNTech, and compared the Nt-Abs levels with anti-SARS-CoV-2 S1/S2 IgGs titres during a 4-month follow-up starting at the first dose of vaccine., (© 2022 Society for Applied Microbiology.)

Sicily was the first Italian region to introduce rotavirus (RV) vaccination with the monovalent G1P[8] vaccine Rotarix® in May 2012. In this study, the seasonal distribution and molecular characterization of RV strains detected over 19 years were compared to understand the effect of Rotarix® on the evolutionary dynamics of human RVs. A total of 7846 stool samples collected from children < 5 years of age, hospitalized with acute gastroenteritis, were tested for RV detection and genotyping. Since 2013, vaccine coverage has progressively increased, while the RV prevalence decreased from 36.1% to 13.3% with a loss of seasonality. The local distribution of RV genotypes changed over the time possibly due to vaccine introduction, with a drastic reduction in G1P[8] strains replaced by common and novel emerging RV strains, such as equine-like G3P[8] in the 2018−2019 season. Comparison of VP7 and VP4 amino acid (aa) sequences with the cognate genes of Rotarix® and RotaTeq® vaccine strains showed specific aa changes in the antigenic epitopes of VP7 and of the VP8* portion of VP4 of the Italian RV strains. Molecular epidemiological surveillance data are required to monitor the emergence of novel RV strains and ascertain if these strains may affect the efficacy of RV vaccines.

Rotavirus A (RVA) is a major etiologic agent of gastroenteritis in children worldwide. Hospital-based surveillance of viral gastroenteritis in paediatric population in Palermo (Italy) from 2017 onwards revealed a sharp increase in G3P[8] RVAs, accounting for 71% of all the RVAs detected in 2019. This pattern had not been observed before in Italy, with G3 RVA usually being detected at rates lower than 3%. In order to investigate this unique epidemiological pattern, the genetic diversity of G3 RVAs identified during a 16-year long surveillance (2004-2019) was explored by systematic sequencing of the VP7 and VP4 genes and by whole genome sequencing of selected G3 strains, representative of the various RVA seasons. Sequence and phylogenetic analyses of the VP7 and VP4 genes revealed the emergence, in 2017 of reassortant equine-like G3P[8], which gradually replaced former G3P[8] strains. The G3P[8] circulating before 2017 showed a Wa-like constellation of genome segments while the G3P[8] that emerged in 2017 had a DS-1-like backbone. On direct inspection of the VP7 and VP4 antigenic epitopes, the equine-like G3P[8] strains possessed several amino acid variations in neutralizing regions compared with vaccine strains. The equine-like G3P[8] RVAs are a further example of the zoonotic impact of animal viruses on human health., (© 2021 Wiley-VCH GmbH.)

Background: The most common Italian rickettsiosis is Mediterranean Spotted Fever (MSF). MSF is commonly associated with a symptom triad consisting of fever, cutaneous rash, and inoculation eschar. The rash is usually maculopapular but, especially in severe presentations, may be petechial. Other typical findings are arthromyalgia and headache. Herein, we describe for the first time an unusual case of Israeli spotted fever (ISF) associated with interstitial pneumonia and pleural effusion in which R. conorii subsp. israelensis was identified by molecular methods in the blood, as well as in the pleural fluid., Case Presentation: A 72-year-old male presented with a 10-day history of remittent fever. On admission, the patient's general condition appeared poor with confusion and drowsiness; the first assessment revealed a temperature of 38.7°, blood pressure of 110/70 mmHg, a blood oxygen saturation level of 80% with rapid, frequent, and superficial breathing using accessory muscles (28 breaths per minute), and an arrhythmia with a heart rate of 90 beats per minute. qSOFA score was 3/3. Chest CT revealed ground-glass pneumonia with massive pleural effusion. Petechial exanthema was present diffusely, including on the palms and soles, and a very little eschar surrounded by a violaceous halo was noted on the dorsum of the right foot. Awaiting the results of blood cultures, broad-spectrum antibiotic therapy with meropenem 1 g q8h, ciprofloxacin 400 mg q12h, and doxycycline 100 mg q12h was initiated. Doxycycline was included in the therapy because of the presence of petechial rash and fever, making us consider a diagnosis of rickettsiosis. This suspicion was confirmed by the positivity of polymerase chain reaction on whole blood for R. conorii subsp. israelensis. Thoracentesis was performed to improve alveolar ventilation. R. conorii subsp. israelensis was again identified in the pleural fluid by PCR technique. On day 4 the clinical condition worsened. Blood exams showed values suggestive of secondary hemophagocytic lymphohistiocytosis; 4 out of 8 diagnostic criteria were present and empirical treatment with prednisone was started resulting in a gradual improvement in general condition., Conclusions: Israeli spotted fever may be a severe disease. A high index of suspicion is required to promptly start life-saving therapy. Pleural effusion and interstitial pneumonia may be part of the clinical picture of severe rickettsial disease and should not lead the physician away from this diagnosis., (© 2021. The Author(s).)

The performance of 2 antigenic commercial assays for enteric adenovirus (AdV) infection, bioNexia Rota-Adeno ImmunoChromatographic Tests (ICT) and LIAISON® Adenovirus ChemiLuminescence Immuno Assays (CLIA), was evaluated on 321 stools from children hospitalized for acute gastroenteritis in Palermo, Italy, using a Real time-PCR (Rt-PCR) as reference method. The CLIA showed higher sensitivity (77% vs 60%), accuracy (94.4 vs 90.9) and concordance (k: 0.81 vs 0.67) with respect to ICT, despite equivalent specificity (98.8%). Using the Ct values of the Rt-PCR as a proxy of the fecal viral load, similar Ct values (mean 9.32 vs 9.89) were observed among the true positive samples, whilst a significant difference (P < 0.05) was observed in false negative samples of CLIA (mean Ct 25.68) and ICT (mean Ct 19.87). Cross-reactivity with other enteric viruses was not observed. These results indicate that both the assays tested are suitable for diagnosis of AdV gastroenteritis., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objectives: In latest years GII.4[P16] and GII.2[P16] noroviruses have become predominant in some temporal/geographical settings. In parallel with the emergence of the GII.P16 polymerase type, norovirus surveillance activity in Italy experienced increasing difficulties in generating sequence data on the RNA polymerase genomic region A, using the widely adopted JV12A/JV13B primer set. Two sets of modified primers (Deg1 and Deg2) were tested in order to improve amplification and typing of the polymerase gene., Methods: Amplification and typing performance of region A primers was assessed in RT-PCR on 452 GII norovirus positive samples obtained from 2194 stool samples collected in 2016-2019 from children hospitalized with acute gastroenteritis., Results: The use of Deg1 increased the rate of samples types in region A from 49.5% to 81.4% and from 21.9% to 69.7% in 2016 and 2017, respectively. The rate of Deg1 typed samples remained high in 2018 (90.1%), but sharply decreased to 11.8% in 2019. The second primers set, Deg2, was able to increase to 64.9% the rate of 2019 samples typed in region A, while typing efficiently 73.2%, 69%, and 86.4% of samples collected in 2016, 2017 and 2018, respectively., Conclusions: The plasticity of norovirus genomes requires continuous updates of the primers used for strain characterization., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2021. Published by Elsevier Ltd.)

Background: Antimicrobial resistance in neonatal intensive care unit (NICU) patients is a threat, due to the frequent use of antimicrobial treatment and invasive devices in fragile babies. Since 2014 an active surveillance program of multidrug-resistant Gram-negative bacteria (MDR-GNB) carriage has been in place in the five NICUs of Palermo, Italy. In 2017 an increase in the prevalence of MDR-GNB, and in particular of extended-spectrum β-lactamases-producing Klebsiella pneumoniae (ESBL-KP), was observed in "Civico" hospital NICU., Aim: To assess the impact of a coordinated intervention strategy in achieving long-lasting reduction of MDR-GNB prevalence in the NICU., Methods: Rectal swabs were obtained monthly and processed to detect MDR-GNB using standard methods. MDR-GNB were characterized by pulsed-field gel electrophoresis (PFGE). Since November 2017 the following intervention measures were applied: (a) two-months intensification of sample collection; (b) stakeholders meetings; (c) improvement of prevention measures and antimicrobial policies., Findings: During the intensified microbiological surveillance MDR-GNB and ESBL-KP were detected in rectal swabs (34.8%; 23.2%), nasal swabs (24.6%; 14.5%), oral swabs (14.5%; 5.4%), milk samples (32.1%; 17.9%), pacifiers swabs (30.8%; 17.9%) and from sub-intensive room surfaces. Thirteen ESBL-KP strains isolated from clinical and environmental samples showed identical PFGE patterns. The prevalence of MDR-GNB and ESBL-KP carriage significantly decreased in the year after intervention compared to the previous year (20.6% vs 62.2%; p < 0.001 and 11.1% vs 57.8%; p < 0.001). MDR-GNB were not detected at all for three months and ESBL-KP for five months. Multivariate analysis of the principal exposure variables showed that admission in the post-intervention period significantly reduced the risk of MDR-GNB carriage (adj-OR = 0.21, 95% CI = 0.076-0.629; p < 0.001)., Conclusions: MDR-GNB broadly circulate in NICU setting, they can colonize different body sites and spread through various vehicles. A coordinated strategy of multiple interventions with active cooperation between epidemiologists and clinicians in the NICU can effectively reduce their circulation and in particular the carriage of the most dangerous ESBL-KP strains.

Background: Acute gastroenteritis is an important cause of childhood morbidity and mortality worldwide. A number of pathogens are responsible for human acute gastroenteritis. The recent introduction of syndromic assays for the diagnosis of enteric infections, including a wide panel of enteric pathogens, has unveiled the frequency of mixed infections. This study was carried out to assess the burden of viral co-infections and the genetic diversity of the viruses detected in children hospitalized with acute gastroenteritis in Italy., Methods: A total of 4161 stool samples collected from diarrheic children over 11 years, from January 2008 to December 2018, were investigated for the presence of four enteric viruses, i.e. group A rotavirus, norovirus, astrovirus and adenovirus. The samples were initially screened by either molecular or immunochromatographic assays and subsequently confirmed by Real-time PCR and sequence analyses., Results: At least one viral agent was detected in 48.6 %of specimens. Rotavirus was the most prevalent virus (24.7 %) followed by norovirus (19.6 %), adenovirus (5.3 %) and astrovirus (3%). Co-infections were detected in 8.3 % of virus-positive patients, with common viral combination being rotavirus with norovirus (70.6 % of co-infections) or with astrovirus (9.6 %). A variety of viral genotypes was detected in co-infections and in single infections. Using Real-time PCR cycle thresholds as a proxy measure of fecal viral load, rotavirus was generally detected at higher levels in co-infected patients., Conclusions: Combining and deciphering measurable indicators of viral load and epidemiological information could be useful for an accurate interpretation of viral co-infections., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Autoantibodies (AAbs) are a hallmark of Type 1 diabetes (T1D). Alterations in the frequency and phenotype of follicular helper (Tfh) T cells have been previously documented in patients with type 1 diabetes (T1D), but the contribution of follicular regulatory T (Treg) cells, which are responsible for suppressing AAb development, is less clear. Here, we investigated the frequency and activation status of follicular (CXCR5 + ) and conventional (CXCR5 - ) Treg cells in the blood of children with new-onset T1D, and children with risk for developing T1D (AAb-positive) and compared them to AAb-negative controls. Blood follicular and conventional Treg cells were higher in frequency in children with new onset T1D, but expressed reduced amounts of PD-1 as compared to AAb-negative children. Interestingly, the proportion of circulating FOXP3 + Tregs expressing PD-1 was also reduced in AAb-positive at-risk children as compared to AAb-negative controls, suggesting its potential use as a biomarker of disease progression. Follicular Treg cells were reduced in frequency in the spleens of prediabetic NOD mice as they became older and turned diabetic. Interestingly, PD-1 expression declined also on circulating follicular and conventional Treg cells in prediabetic NOD mice as they aged. Together, these findings show that the frequency of circulating follicular and conventional Treg cells and their levels of PD-1 change with disease progression in children at-risk for developing T1D and in NOD mice., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aims: To evaluate physicians' adjustments of insulin pump settings based on continuous glucose monitoring (CGM) for patients with type 1 diabetes and to compare these to automated insulin dose adjustments., Methods: A total of 26 physicians from 16 centres in Europe, Israel and South America participated in the study. All were asked to adjust insulin dosing based on insulin pump, CGM and glucometer downloads of 15 patients (mean age 16.2 ± 4.3 years, six female, mean glycated haemoglobin 8.3 ± 0.9% [66.8 ± 7.3 mmol/mol]) gathered over a 3-week period. Recommendations were compared for the relative changes in the basal, carbohydrate to insulin ratio (CR) and correction factor (CF) plans among physicians and among centres and also between the physicians and an automated algorithm, the Advisor Pro (DreaMed Diabetes Ltd, Petah Tikva, Israel). Study endpoints were the percentage of comparison points for which there was full agreement on the trend of insulin dose adjustments (same trend), partial agreement (increase/decrease vs no change) and full disagreement (opposite trend)., Results: The percentages for full agreement between physicians on the trend of insulin adjustments of the basal, CR and CF plans were 41 ± 9%, 45 ± 11% and 45.5 ± 13%, and for complete disagreement they were 12 ± 7%, 9.5 ± 7% and 10 ± 8%, respectively. Significantly similar results were found between the physicians and the automated algorithm. The algorithm magnitude of insulin dose change was at least equal to or less than that proposed by the physicians., Conclusions: Physicians provide different insulin dose recommendations based on the same datasets. The automated advice of the Advisor Pro did not differ significantly from the advice given by the physicians in the direction or magnitude of the insulin dosing., (© 2018 John Wiley & Sons Ltd.)

Mediterranean spotted fever (MSF) is endemic in Italy, where Rickettsia conorii subsp. conorii was thought to be the only pathogenic rickettsia and Rhipicephalus sanguineus the vector and main reservoir. R. conorii subsp. israelensis, which belongs to the R. conorii complex, is the agent of Israeli spotted fever (ISF); apart from Israel, it has also been found in Italy (Sicily and Sardinia) and in different regions of Portugal. We describe here two severe cases of ISF which occurred in otherwise healthy Italian adults. Their characteristics are analyzed and discussed in the light of other 91 cases found through a systematic review of international literature., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The global spread of Klebsiella pneumoniae producing Klebsiella pneumoniae carbapenemase (KPC) has been mainly associated with the dissemination of high-risk clones. In the last decade, hospital outbreaks involving KPC-producing K. pneumoniae have been predominantly attributed to isolates belonging to clonal group (CG) 258. However, results of recent epidemiological analysis indicate that KPC-producing sequence type (ST) 307, is emerging in different parts of the world and is a candidate to become a prevalent high-risk clone in the near future. Here we show that the ST307 genome encodes genetic features that may provide an advantage in adaptation to the hospital environment and the human host. Sequence analysis revealed novel plasmid-located virulence factors, including a cluster for glycogen synthesis. Glycogen production is considered to be one of the possible adaptive responses to long-term survival and growth in environments outside the host. Chromosomally-encoded virulence traits in the clone comprised fimbriae, an integrative conjugative element carrying the yersiniabactin siderophore, and two different capsular loci. Compared with the ST258 clone, capsulated ST307 isolates showed higher resistance to complement-mediated killing. The acquired genetic features identified in the genome of this new emerging clone may contribute to increased persistence of ST307 in the hospital environment and shed light on its potential epidemiological success.

Genetic mechanisms of methicillin resistance are still relevant in staphylococci. The aims of this study are to assess the possible exchanges of staphylococcal cassette chromosome mec (SCCmec) among isolates of methicillin-resistant staphylococci (MRS) and to check for known or new mutations in mecA DNA. A total of 35 MRS non-repetitive isolates were recovered, including 20 Staphylococcushaemolyticus, 7 Staphylococcusaureus, 4 Staphylococcussciuri, 2 Staphylococcussaprophyticus and 1 isolate each of Staphylococcusxylosus and Staphylococcuslentus. Only 16 of the 35 strains were assigned to known SCCmec types: 7 SCCmec VII, 6 SCCmec IV and 3 SCCmec III, with possible horizontal transfer of the SCCmec VII from methicillin-resistant S. haemolyticus to methicillin-susceptible S. aureus. mecA gene sequencing in ten selected isolates allowed description of nine punctual mutations, seven of which were reported for the first time. The most frequent mutation was G246E, identified in isolates of methicillin-resistant S. aureus, S. sciuri, S. saprophyticus and S. lentus. These results emphasized the high degree of genetic diversity of SCCmec element in MRS and describe new missense mutations in mecA, which might be important in understanding the evolution of methicillin and new β-lactam resistance.

Extraintestinal pathogenic Escherichia coli sequence type 131 (ST131), typically fluoroquinolone-resistant (FQ-R) and/or extended-spectrum β-lactamase (ESBL)-producing, has emerged globally. Among clinical isolates, ST131, primarily its H30-R and H30-Rx subclones, accounts for most antimicrobial-resistant E. coli and is the dominant E. coli strain worldwide. We assessed its prevalence and characteristics among raw chicken meat samples on sale in Palermo, Italy. A collection of 237 fluoroquinolone resistant and ESBL/AmpC producing E. coli isolates, which had been isolated from processed retail chicken meat in the period May 2013-April 2015, was analyzed. Established polymerase chain reaction methods were used to define ST131 and its H30 subclones, ESBL, AmpC, and plasmid-mediated quinolone resistance (PMQR) determinants. Amplified Fragment Length Polymorphism (AFLP) was performed to assess the relatedness among ST131 isolates. Out of the 237 E. coli isolates, 12 isolates belonged to the phylogenetic group B2. Based on the molecular definition of ExPEC, all isolates were attributed with the status of ExPEC. SNP-PCR results confirmed that nine isolates were ST131. SNP-PCR for H30-R and H30-Rx subclones showed that six and three ExPEC ST131 were positive for H30-R and H30-Rx, respectively. The results of AFLP showed that, except for four isolates grouped into two clusters which proved to be indistinguishable, the isolates under study were genetically heterogeneous. To the best of our knowledge, this is the first report of H30-R and H30-Rx subclones in animal food samples. Our findings appear to support the role of food chain in their transmission to humans., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objectives: We performed a 1-year prospective surveillance study on MRSA colonization within the five NICUs of the metropolitan area of Palermo, Italy. The purpose of the study was to assess epidemiology of MRSA in NICU from a network perspective., Methods: Transfer of patients between NICUs during 2014 was traced based on the annual hospital discharge records. In the period February 2014-January 2015, in the NICU B, at the University teaching hospital, nasal swabs from all infants were collected weekly, whereas in the other four NICUs (A, C, D, E) at 4 week-intervals of time. MRSA isolates were submitted to antibiotic susceptibility testing, SCCmec typing, PCR to detect lukS-PV and lukF-PV (lukS/F-PV) genes and the gene encoding the toxic shock syndrome toxin (TSST-1), multilocus variable number tandem repeat fingerprinting (MLVF), and multilocus sequence typing (MLST)., Results: In the period under study, 587 nasal swabs were obtained from NICU B, whereas 218, 180, 157, and 95 from NICUs A, C, D, and E, respectively. Two groups of NICUs at high prevalence and low prevalence of MRSA colonization were recognized. Overall, 113 isolates of MRSA were identified from 102 infants. Six MLVF types (A-F) were detected, with type C being subdivided into five subtypes. Five sequence types (STs) were found with ST22-IVa being the most frequent type in all NICUs. All the MRSA molecular subtypes, except for ST1-IVa, were identified in NICU B., Conclusions: Our findings support the need to approach surveillance and infection control in NICU in a network perspective, prioritizing referral healthcare facilities.

Klebsiella pneumoniaesequence type (ST) 307, carryingblaKPC-3,blaCTX-M-15,blaOXA-1,aac(6')-Ib-cr, andqnrB1 genes, is replacing the predominant hyperepidemic ST258 clone in Italy. Whole-genome and complete plasmid sequencing of one ST307 strain was performed and new features were identified., (Copyright © 2016 Villa et al.)

Colonization and infection by multidrug-resistant gram-negative bacilli (MDR GNB) in neonatal intensive care units (NICUs) are increasingly reported.We conducted a 5-year prospective cohort surveillance study in a tertiary NICU of the hospital "Paolo Giaccone," Palermo, Italy. Our objectives were to describe incidence and trends of MDR GNB colonization and the characteristics of the most prevalent organisms and to identify the risk factors for colonization. Demographic, clinical, and microbiological data were prospectively collected. Active surveillance cultures (ASCs) were obtained weekly. Clusters of colonization by extended spectrum β-lactamase (ESBL) producing Escherichia coli and Klebsiella pneumoniae were analyzed by conventional and molecular epidemiological tools.During the study period, 1152 infants were enrolled in the study. Prevalences of colonization by MDR GNB, ESBL-producing GNB and multiple species/genera averaged, respectively, 28.8%, 11.7%, and 3.7%. Prevalence and incidence density of colonization by MDR GNB and ESBL-producing GNB showed an upward trend through the surveillance period. Rates of ESBL-producing E coli and K pneumoniae colonization showed wide fluctuations peaking over the last 2 years. The only independent variables associated with colonization by MDR GNB and ESBL-producing organisms and multiple colonization were, respectively, the days of NICU stay (odds ratio [OR] 1.041), the days of exposure to ampicillin-sulbactam (OR 1.040), and the days of formula feeding (OR 1.031). Most clusters of E coli and K pneumoniae colonization were associated with different lineages. Ten out of 12 clusters had an outborn infant as their index case.Our study confirms that MDR GNB are an increasing challenge to NICUs. The universal once-a-week approach allowed us to understand the epidemiology of MDR GNB, to timely detect new clones and institute contact precautions, and to assess risk factors. Collection of these data can be an important tool to optimize antimicrobials use and control the emergence and dissemination of resistances in NICU., Competing Interests: The authors have no funding and conflicts of interest to disclose.

A carbapenem-resistant sequence type 512 (ST512) Klebsiella pneumoniae carbapenemase 3 (KPC-3)-producing K. pneumoniae strain showing a novel variant plasmid content was isolated in Palermo, Italy, in 2014. ST512 is a worldwide successful clone associated with the spread of bla(KPC) genes located on the IncFIIk pKpQIL plasmid. In our ST512 strain, the bla(KPC-3) gene was unusually located on an IncX3 plasmid, whose complete sequence was determined. Two copies of bla(KPC-3)::Tn4401a caused by intramolecular transposition events were detected in the plasmid., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The resistance of Helicobacter pylori strains to clarithromycin is increasing in several developed countries and their association with a genetic pattern circulation has been variously explained as related to different geographical areas. In this study we have reported: the prevalence of the resistance of H. pylori, isolated in Sicily, to clarithromycin; the principal point of mutation associated with this resistance; and the more frequent association between resistance to clarithromycin and cagA, the EPIYA motif, and the vacA and oipA genes. Resistance to clarithromycin was detected in 25% of cases, the main genetic mutation involved being A2143G. The cagA gene was present in 48% of cases and the distribution of the EPIYA motif was: ABC in 35 cases; ABCC in 8 cases; ABCCC in 2 cases; ABC-ABCC in 2 cases; and ABC-ABCC-ABCCC in 1 case. Regarding the vacA allele, an s1i1m1 combination was detected in 35% of cases, s1i1m2 in 12 %, s1i2m2 in 12%, s2i2m2 in 40%, and a double s1m1-m2 mosaic in 1% of cases. The status of the oipA gene was 'off' in 45% of cases and 'on' in 55%. Resistance to clarithromycin was found to be high in Sicily, but no correlation was found among resistance to clarithromycin, the vacA gene and oipA status; a higher correlation was observed between resistant strains and cagA-negative strains.

Background: In Italy, Klebsiella pneumoniae carbapenemase producing K. pneumoniae (KPC-Kp) strains are highly endemic and KPC producing CC258 is reported as the widely predominating clone. In Palermo, Italy, previous reports have confirmed this pattern. However, recent preliminary findings suggest that an epidemiological change is likely ongoing towards a polyclonal KPC-Kp spread. Here we present the results of molecular typing of 94 carbapenem non susceptible K. pneumoniae isolates detected during 2014 in the three different hospitals in Palermo, Italy., Methods and Results: Ninety-four consecutive, non replicate carbapenem non susceptible isolates were identified in the three largest acute general hospitals in Palermo, Italy, in the six-month period March-August 2014. They were characterized by PCR for β-lactam, aminoglycoside and plasmid mediated fluoroquinolone resistance genetic determinants. The mgrB gene of the colistin resistant isolates was amplified and sequenced. Clonality was assessed by pulsed field gel electrophoresis and multilocus sequence typing. Eight non-CC258 sequence types (STs) were identified accounting for 60% of isolates. In particular, ST307 and ST273 accounted for 29% and 18% of isolates. CC258 isolates were more frequently susceptible to gentamicin and non-CC258 isolates to amikacin. Colistin non susceptibility was found in 42% of isolates. Modifications of mgrB were found in 32 isolates., Conclusions: Concurrent clonal expansion of some STs and lateral transmission of genetic resistance determinants are likely producing a thorough change of the KPC-Kp epidemiology in Palermo, Italy. In our setting mgrB inactivation proved to substantially contribute to colistin resistance. Our findings suggest the need to continuously monitor the KPC-Kp epidemiology and to assess by a nationwide survey the possible shifting towards a polyclonal epidemic.

Background: Globally, antimicrobial drug-resistant Escherichia coli is among the most common etiological agents of invasive disease in humans. In Europe, increasing proportions of infections due to third-generation cephalosporins and/or fluoroquinolone-resistant extraintestinal pathogenic E. coli (ExPEC) strains are reported. E. coli from poultry are those more closely linked to human E. coli, but lack of reliable data makes it difficult to assess the attributable risk of different food sources. In the present study, our objective was to investigate the antimicrobial resistance profile, phylogenetic background, and virulence factors of E. coli isolates from broiler chicken meat sold at retail in Palermo, Italy., Materials and Methods: Isolation of multidrug resistant (MDR) E. coli was performed during April-December 2013 on a total of 163 chicken meat samples. Susceptibility to a panel of nine antimicrobial agents was determined. PCR assays were carried out to detect extended-spectrum β-lactamase (ESBL), plasmid-mediated AmpC β-lactamase, and plasmid-mediated quinolone resistance (PMQR) genes, phylogenetic group, and ExPEC-associated traits. A single nucleotide polymorphism (SNP) PCR was done to detect E. coli sequence type (ST)131., Results: One hundred thirty-four isolates from 109 meat samples were MDR. B1 was the most prevalent phylogenetic group (47.8%), followed by groups D (25.4%), A (22.3%), and B2 (4.5%). ESBLs and AmpC β-lactamases were detected by PCR in 132 (98.5%) and 15 (11.2%) isolates. PMQR determinants were detected in 122 (91%) isolates. Twenty-two MDR isolates met the molecular definition of ExPEC. SNP-PCR results confirmed that four B2 isolates were ST131. Enterobacterial Repetitive Intergenic Consensus sequence-PCR analysis showed a large heterogeneity with 55 unique profiles and 31 clusters including 2-4 isolates., Conclusions: An alarmingly high prevalence of MDR E. coli from broiler chicken meat is evident in our geographic area. The ongoing use of antimicrobial drugs in livestock should be urgently restricted, particularly in the poultry sector.