First imported case of Candida auris infection in Milan, Italy: genomic characterisation.

PURPOSE: Candida auris, an emerging multidrug-resistant yeast, has been reported worldwide. In Italy, the first case was reported in 2019. We describe the first case of C. auris, imported from Greece, in Milan, using whole genome sequencing to characterise mutations associated with antifungal resistance. CASE PRESENTATION: On October 2022 an 80-year-old Italian man was hospitalised in Greece. In the absence of clinical improvement, the patient was transferred to our hospital, in Italy, where blood culture resulted positive for C. auris. Despite therapy, the patient died of septic shock. In a phylogenetic analysis the genome was assigned to Clade I with strains from Kenya, United Arab Emirates and India. D1/D2 region resulted identical to a Greek strain, as for many other strains from different World regions, highlighting the diffusion of this strain. CONCLUSION: Importation of C. auris from abroad has been previously described. We report the first case of C. auris imported into Italy from Greece, according to phylogenetic analysis. This case reinforces the need for monitoring critically ill hospitalised patients also for fungi and addresses the need for the standardisation of susceptibility testing and strategies for diagnosis and therapy.

Risk factors associated with bacteremia in COVID-19 patients admitted to intensive care unit: a retrospective multicenter cohort study.

PURPOSE: This multicenter observational study was done to evaluate risk factors related to the development of BSI in patients admitted to ICU for COVID-19. METHODS: All patients with COVID-19 admitted in two COVID-19 dedicated ICUs in two different hospital between 02-2020 and 02-2021 were recruited. RESULT: 537 patients were included of whom 265 (49.3%) experienced at least one BSI. Patients who developed bacteremia had a higher SOFA score [10 (8-12) vs 9 (7-10), p < 0.001], had been intubated more frequently [95.8% vs 75%, p < 0.001] and for a median longer time [16 days (9-25) vs 8 days (5-14), p < 0.001]. Patients with BSI had a median longer ICU stay [18 days (12-31.5) vs 9 days (5-15), p < 0.001] and higher mortality [54% vs 42.3%, p < 0.001] than those who did not develop it. Development of BSI resulted in a higher SOFA score [aHR 1.08 (95% CI 1.03-1.12)] and a higher Charlson score [csAHR 1.15 (95% CI 1.05-1.25)]. CONCLUSION: A high SOFA score and a high Charlson score resulted associated with BSI's development. Conversely, immunosuppressive therapy like steroids and tocilizumab, has no role in increasing the risk of bacteremia.

SARS-CoV-2 in the knee joint: a cadaver study.

OBJECTIVES: Despite the considerable research efforts being made to learn more about COVID-19, little is known about the presence of SARS-CoV-2 genetic material in biological fluids other than respiratory droplets, blood, and feces. The aim of this post-mortem study was to assess the presence of SARS-CoV-2 RNA in the knee synovial fluid, synovial tissue, and bone tissue of COVID-19 patients in order to discover whether the joint is a possible route of transmission during orthopaedic surgical procedures, and clarify the possible role of SARS-CoV-2 as a directly arthritogenic virus. METHODS: Post-mortem synovial fluid, synovial tissue and bone tissue samples were collected from the knees of five patients who died of COVID-19 in our hospital between September and October 2020, and analysed for the presence of SARS-CoV-2 using a commercial real-time polymerase chain reaction (RT-PCR) panel. Quantitative RT-PCR was used to test post-mortem nasopharyngeal swabs of all of the patients. RESULTS: No SARS-CoV-2 RNA was detected in any of the knee samples, despite the positivity of the throat swab. CONCLUSIONS: Our findings indicate that SARS-CoV-2 was not detected in knee synovial fluid, synovial membrane or bone. This makes it unlikely that these are potential sources of contagion, and suggests that SARS-CoV-2 is not directly arthritogenic.

Psychometrics and diagnostics of Italian cognitive screening tests: a systematic review.

BACKGROUND: Cognitive screening tests (CSTs) are crucial to neuropsychological diagnostics, and thus need to be featured by robust psychometric and diagnostic properties. However, CSTs happen not to meet desirable statistical standards, negatively affecting their level of recommendations and applicability. This study aimed at (a) providing an up-to-date compendium of available CSTs in Italy, (b) report their psychometric and diagnostic properties, and (c) address related limitations. METHODS: This review was implemented by consulting Preferred Reporting Items for Systematic Reviews and Meta-Analyses and pre-registered on the International Prospective Register of Systematic Reviews. Standardization and usability studies focusing on norms, validity, reliability, or sensitivity/specificity (and derived metrics) in adults were considered for eligibility. Quality assessment was performed by means of an ad hoc checklist collecting information on sampling, psychometrics/diagnostics, norming, and feasibility. RESULTS: Sixty studies were included out of an initial N = 683. Identified CSTs (N = 40) were classified into general, domain-, and disease-specific (N = 17, 7, and 16, respectively), the latter being less statistically robust than remaining categories. Validity and reliability evidence was provided for 29 and 26 CSTs, respectively, sensitivity/specificity for 20 and norms for 33. Prevalence- and post-test-based diagnostic metrics were seldomly represented; factorial structures, ceiling/floor effects, and acceptability rarely investigated; content, face, and ecological validity never assessed. DISCUSSION: Although available Italian CSTs overall met basic psychometric/diagnostic requirements, their statistical profile often proved to be poor on several properties that are desirable for clinical applications, with a few exceptions among general and domain-specific ones.

Geographical reconstruction of the SARS-CoV-2 outbreak in Lombardy (Italy) during the early phase.

The first identification of autochthonous transmission of SARS-CoV-2 in Italy was documented by the Laboratory of Clinical Microbiology, Virology and Bioemergencies of L. Sacco Hospital (Milano, Italy) on 20th February 2020 in a 38 years old male patient, who was found positive for pneumonia at the Codogno Hospital. Thereafter Lombardy has reported the highest prevalence of COVID-19 cases in the country, especially in Milano, Brescia and Bergamo provinces. The aim of this study was to assess the potential presence of different viral clusters belonging to the six main provinces involved in Lombardy COVID-19 cases in order to highlight peculiar province-dependent viral characteristics. A phylogenetic analysis was conducted on 20 full length genomes obtained from patients addressing to several Lombard hospitals from February 20th to April 4th, 2020, aligned with 41 Italian viral genome assemblies available on GISAID database as of 30th March, 2020: two main monophyletic clades, containing 8 and 53 isolates, respectively, were identified. Noteworthy, Bergamo isolates mapped inside the small clade harbouring M gene D3G mutation. The molecular clock analysis estimated a cluster divergence approximately one month before the first patient identification, supporting the hypothesis that different SARS-CoV-2 strains had spread worldwide at different times, but their presence became evident only in late February along with Italian epidemic emergence. Therefore, this epidemiological reconstruction suggests that virus initial circulation in Lombardy was ascribable to multiple introduction. The phylogenetic reconstruction robustness, however, will be improved when more genomic sequences are available, in order to guarantee a complete epidemiological surveillance.

Eradication of a Multidrug-Resistant, Carbapenemase-Producing Klebsiella pneumoniae Isolate Following Oral and Intra-rectal Therapy With a Custom Made, Lytic Bacteriophage Preparation.

In July 2017, a patient presented colonization with a multidrug-resistant, carbapenemase (KPC-3)-producing Klebsiella pneumoniae isolate. A custom-made, lytic bacteriophage preparation was administered to the patient in December 2017, with subsequent eradication of the microorganism and without adverse effects.

Microbial biofilm correlates with an increased antibiotic tolerance and poor therapeutic outcome in infective endocarditis.

BACKGROUND: Infective endocarditis (IE) is associated with high rates of mortality. Prolonged treatments with high-dose intravenous antibiotics often fail to eradicate the infection, frequently leading to high-risk surgical intervention. By providing a mechanism of antibiotic tolerance, which escapes conventional antibiotic susceptibility profiling, microbial biofilm represents a key diagnostic and therapeutic challenge for clinicians. This study aims at assessing a rapid biofilm identification assay and a targeted antimicrobial susceptibility profile of biofilm-growing bacteria in patients with IE, which were unresponsive to antibiotic therapy. RESULTS: Staphylococcus aureus was the most common isolate (50%), followed by Enterococcus faecalis (25%) and Streptococcus gallolyticus (25%). All microbial isolates were found to be capable of producing large, structured biofilms in vitro. As expected, antibiotic treatment either administered on the basis of antibiogram or chosen empirically among those considered first-line antibiotics for IE, including ceftriaxone, daptomycin, tigecycline and vancomycin, was not effective at eradicating biofilm-growing bacteria. Conversely, antimicrobial susceptibility profile of biofilm-growing bacteria indicated that teicoplanin, oxacillin and fusidic acid were most effective against S. aureus biofilm, while ampicillin was the most active against S. gallolyticus and E. faecalis biofilm, respectively. CONCLUSIONS: This study indicates that biofilm-producing bacteria, from surgically treated IE, display a high tolerance to antibiotics, which is undetected by conventional antibiograms. The rapid identification and antimicrobial tolerance profiling of biofilm-growing bacteria in IE can provide key information for both antimicrobial therapy and prevention strategies.

Genomic Characterization of an ST1153 PVL-producing Methicillin Resistant Staphylococcus aureus Clinical Isolate in Italy.

Methicillin-resistant Staphylococcus aureus (MRSA) clones are rapidly increasing beyond the hospital into the community, livestock farming and environmental settings. An Italian man, a professional diver working in Egypt, was admitted to Infectious Diseases Clinic-ASST Fatebenefratelli Sacco for ulcerative skin lesions. An MRSA strain was isolated from the lesions' purulent exudate and the nasal colonization was also ascertained. The strain, characterized by whole genome sequencing, resulted to be Panton-Valentine Leukocidin (PVL) positive, SCCmecI - spa-type t504, and belonging to the sequence type 1153, sporadically described worldwide.

Detection of ST1702 Escherichia coli blaNDM-5 and blaCMY-42 genes positive isolates from a Northern Italian hospital.

We describe two multi drug-resistant (MDR) carbapenemase-producing Escherichia coli clinical isolates from an acute hospital in Milan. Both strains, isolated from a surgical wound sample and a surveillance rectal swab respectively, were positive for a blaNDM-type gene by Xpert Carba-R test. The whole-genome sequence characterization disclosed several resistance determinants: blaNDM-5, blaCMY-42, blaTEM-198, rmtB, mphA. The two isolates belonged to phylogenetic group A, sequence type (ST) 1702 and serotype O89:H9. PCR-based replicon typing and conjugation assay demonstrated an IncI1 plasmid localization for both blaNDM-5 and blaCMY-42 genes. This is the first report of a ST1702 NDM-5 and CMY-42- producing E. coli clone in Italy.

Whole genome sequencing for the molecular characterization of carbapenem-resistant Klebsiella pneumoniae strains isolated at the Italian ASST Fatebenefratelli Sacco Hospital, 2012-2014.

BACKGROUND: The emergence of carbapenem-resistant Klebsiella pneumoniae strains is threatening antimicrobial treatment. METHODS: Sixty-eight carbapenemase-producing K. pneumoniae strains isolated at Luigi Sacco University Hospital-ASST Fatebenefratelli Sacco (Milan, Italy) between 2012 and 2014 were characterised microbiologically and molecularly. They were tested for drug susceptibility and carbapenemase phenotypes, investigated by means of repetitive extra-genic palindromic polymerase chain reaction (REP-PCR), and fully sequenced by means of next-generation sequencing for the in silico analysis of multi-locus sequence typing (MLST), their resistome, virulome and plasmid content, and their core single nucleotide polymorphism (SNP) genotypes. RESULTS: All of the samples were resistant to carbapenems, other ß-lactams and ciprofloxacin; many were resistant to aminoglycosides and tigecycline; and seven were resistant to colistin. Resistome analysis revealed the presence of blaKPC genes and, less frequently blaSHV, blaTEM, blaCTX-M and blaOXA, which are related to resistance to carbapenem and other ß-lactams. Other genes conferring resistance to aminoglycoside, fluoroquinolone, phenicol, sulphonamide, tetracycline, trimethoprim and macrolide-lincosamide-streptogramin were also detected. Genes related to AcrAB-TolC efflux pump-dependent and pump-independent tigecycline resistance mechanisms were investigated, but it was not possible to clearly correlate the genomic features with tigecycline resistance because of the presence of a common mutation in susceptible, intermediate and resistant strains. Concerning colistin resistance, the mgrB gene was disrupted by an IS5-like element, and the mobile mcr-1 and mcr-2 genes were not detected in two cases. The virulome profile revealed type-3 fimbriae and iron uptake system genes, which are important during the colonisation stage in the mammalian host environment. The in silico detected plasmid replicons were classified as IncFIB(pQil), IncFIB(K), ColRNAI, IncX1, IncX3, IncFII(K), IncN, IncL/M(pMU407) and IncFIA(HI1). REP-PCR showed five major clusters, and MLST revealed six different sequence types: 512, 258, 307, 1519, 745 and 101. Core SNP genotyping, which led to four clusters, correlated with the MLST data. Isolates of the same sequencing type often had common genetic traits, but the SNP analysis allowed greater strain tracking and discrimination than either the REP-PCR or MLST analysis. CONCLUSION: Our findings support the importance of implementing bacterial genomics in clinical medicine in order to complement traditional methods and overcome their limited resolution.

Pediatric Lyme Disease in Northern Italy: An 18-Year Single-Center Case Series.

Tracing the profile of pediatric Lyme borreliosis (LB) in Europe is difficult due to the interregional variation in its incidence and lack in notifications. Moreover, the identification of LB can be challenging. This study is an 18-year case series of 130 children and adolescents aged under 18 years referred to the Pediatric Infectious Diseases Unit at L. Sacco Hospital, Milan, with suspicion of LB, between January 2005 and July 2023. The routine serological workup consisted of a two-step process: an initial screening test followed by Western blot (WB). Forty-four (34%) patients were diagnosed with LB. The median age was six years, and 45% were females. Of the children with erythema migrans (EM), 33 (57%) were confirmed as having true EM, and, of these, 4 (12%) were atypical. Ten (23%) patients had early disseminated/late diseases, including facial nerve palsy (n = 3), early neuroborreliosis (n = 1), arthritis (n = 3), relapsing fever (n = 2), and borrelial lymphocytoma (n = 1). No asymptomatic infections were documented. Over seventy percent of confirmed LB cases (n = 31/44) recalled a history of tick bites; in this latter group, 19 (61%) were from the area of the Po River valley in Lombardy. Almost half of the children evaluated for LB complained of non-specific symptoms (fatigue, musculoskeletal pain, skin lesions/rash, and persistent headache), but these symptoms were observed in only two patients with confirmed LB. Most LB cases in our study were associated with EM; two-tier testing specificity was high, but we found frequent non-adherence to international recommendations with regard to the timing of serology, application of the two-step algorithm, and antibiotic over-prescription. Most children were initially assessed for a tick bite or a skin lesion suggestive of EM by a family pediatrician, highlighting the importance of improving awareness and knowledge around LB management at the primary healthcare level. Finally, the strengthening of LB surveillance at the national and European levels is necessary.

Genetic barriers more than environmental associations explain Serratia marcescens population structure.

Bacterial species often comprise well-separated lineages, likely emerged and maintained by genetic isolation and/or ecological divergence. How these two evolutionary actors interact in the shaping of bacterial population structure is currently not fully understood. In this study, we investigate the genetic and ecological drivers underlying the evolution of Serratia marcescens, an opportunistic pathogen with high genomic flexibility and able to colonise diverse environments. Comparative genomic analyses reveal a population structure composed of five deeply-demarcated genetic clusters with open pan-genome but limited inter-cluster gene flow, partially explained by Restriction-Modification (R-M) systems incompatibility. Furthermore, a large-scale research on hundred-thousands metagenomic datasets reveals only a partial habitat separation of the clusters. Globally, two clusters only show a separate gene composition coherent with ecological adaptations. These results suggest that genetic isolation has preceded ecological adaptations in the shaping of the species diversity, an evolutionary scenario coherent with the Evolutionary Extended Synthesis.

Cerebral Infectious Opportunistic Lesions in a Patient with Acute Myeloid Leukaemia: The Challenge of Diagnosis and Clinical Management.

Central nervous system (CNS) lesions, especially invasive fungal diseases (IFDs), in immunocompromised patients pose a great challenge in diagnosis and treatment. We report the case of a 48-year-old man with acute myeloid leukaemia and probable pulmonary aspergillosis, who developed hyposthenia of the left upper limb, after achieving leukaemia remission and while on voriconazole. Magnetic resonance imaging (MRI) showed oedematous CNS lesions with a haemorrhagic component in the right hemisphere with lepto-meningitis. After 2 weeks of antibiotics and amphotericin-B, brain biopsy revealed chronic inflammation with abscess and necrosis, while cultures were negative. Clinical recovery was attained, he was discharged on isavuconazole and allogeneic transplant was postponed, introducing azacitidine as a maintenance therapy. After initial improvement, MRI worsened; brain biopsy was repeated, showing similar histology; and 16S metagenomics sequencing analysis was positive (Veilonella, Pseudomonas). Despite 1 month of meropenem, MRI did not improve. The computer tomography and PET scan excluded extra-cranial infectious-inflammatory sites, and auto-immune genesis (sarcoidosis, histiocytosis, CNS vasculitis) was deemed unlikely due to the histological findings and unilateral lesions. We hypothesised possible IFD with peri-lesion inflammation and methyl-prednisolone was successfully introduced. Steroid tapering is ongoing and isavuconazole discontinuation is planned with close follow-up. In conclusion, the management of CNS complications in immunocompromised patients needs an interdisciplinary approach.

Cultivation and sequencing-free protocol for Serratia marcescens detection and typing.

Serratia marcescens is an opportunistic pathogen that survives in inhospitable environments causing large outbreaks, particularly in neonatal intensive care units (NICUs). Genomic studies revealed that most S. marcescens nosocomial infections are caused by a specific clone (here "Infectious clone"). Whole genome sequencing (WGS) is the only portable method able to identify this clone, but it requires days to obtain results. We present a cultivation-free hypervariable-locus melting typing (HLMT) protocol for the fast detection and typing of S. marcescens, with 100% detection capability on mixed samples and a limit of detection that can reach the 10 genome copies. The protocol was able to identify the S. marcescens infectious clone with 97% specificity and 96% sensitivity when compared to WGS, yielding typing results portable among laboratories. The protocol is a cost and time saving method for S. marcescens detection and typing for large environmental/clinical surveillance screenings, also in low-middle income countries.

Respiratory syncytial virus burden and risk factors for severe disease in patients presenting to the emergency department with flu-like symptoms or acute respiratory failure.

BACKGROUND: Predictors of Respiratory Syncytial Virus (RSV) infection and determinants of RSV unfavorable outcomes are still unclear. We assessed RSV burden and investigated the risk factors associated with RSV positive swab and RSV severe disease. METHODS: A retrospective, single center, cohort study included all consecutive patients referred to the emergency department of L. Sacco University Hospital (Milan) with flu-like symptoms or acute respiratory failure (aRF) tested per protocol for SARS-CoV-2, RSV, Influenza A (InvA) during the 2022-2023 autumn/winter season. Clinical characteristics and patients' outcomes were registered. Respiratory failure, need for respiratory support, shock, sepsis or in-hospital death defined severe disease. MAIN FINDINGS: The analysis included 717 patients (65.1% negative swab, 14.1% InvA, 8.5% RSV, 8.6% SARS-CoV-2, 3.6% other viruses). Compared with the study cohort, RSV patients had the highest occurrence of aRF (62.7%) and severe disease (70.5%); mortality was similar to InvA (6.6% vs 5.9%, p = 0.874). Compared with InvA patients, RSV patients were older (p = 0.009), had higher Charlson index (p = 0.001), higher prevalence of chronic heart failure (p = 0.001) and were more frequently on ICS (p = 0.026) and immunosuppressants (p = 0.018). Heart failure [OR (95%CI):3.286 (1.031-10.835); p = 0.041], chronic exposure to ICS [OR (95%CI):2.377 (1.254-4.505); p = 0.008] and immunosuppressants [OR (95%CI):3.661 (1.246-10.754); p = 0.018] predicted RSV infection. Glycaemia ≥120 mg/dL [OR (95%CI):5.839 (1.155-29.519); p = 0.033], leucocytes ≥8000 cells/µL [OR (95%CI):5.929 (1.090-32.268); p = 0.039], and past/active smoking [OR (95%CI):7.347 (1.301-41.500); p = 0.024] predicted severe RSV disease. CONCLUSIONS: RSV infection is associated with significant mortality and morbidity. Preventive strategies for RSV infection such as vaccination are highly warranted, especially in older patients with cardiovascular and chronic respiratory conditions.

The history of Lyme disease in Italy and its spread in the Italian territory.

Lyme borreliosis (LB) is the most common vector-borne zoonotic inflammatory disease in the Northern Hemisphere. In Italy, the first case was diagnosed in 1985 in a woman in Liguria, while the second, in 1986 in Friuli-Venezia Giulia, documenting the infection in northern Italy. Both diagnoses were confirmed by serological assessment by an indirect immunofluorescence (IFI) technique. Borrelia cultivation from both Ixodes ricinus ticks and human lesions in Trieste (Friuli-Venezia Giulia) identified Borrelia afzelii as the prevalent genospecies; nevertheless, Borrelia garinii, Borrelia burgdorferi (sensu stricto), and Borrelia valaisiana (VS116 Group) were also detected, although less frequently. LB was also documented in other Italian regions: in Tuscany (1991), Trentino-Alto Adige (1995-1996), Emilia-Romagna (1998), Abruzzo (1998), and more recently, Lombardy. Nevertheless, data on LB in other Italian regions, especially in southern Italy and islands, are poor. The aim of this study is to document the spread of LB in Italy through the collection of data from LB patients in eight Italian hospitals located in different Italian regions. Diagnostic criteria for LB diagnosis are as follows: i) the presence of erythema migrans (EM) or ii) a clinical picture suggestive of LB, confirmed by serological tests and/or PCR positivity for Borrelia detection. In addition, data also included the place of residence (town and region) and the place where patients became infected. During the observation period, 1,260 cases were gathered from the participating centers. Although different in extent from northern Italy to central/southern Italy, this study shows that LB is widespread throughout Italy.