Valeria Fox

Background/Objectives: This study aimed to characterize the epidemiology, resistance, virulence determinants, and genomic relatedness of OXA-48-producing Klebsiella pneumoniae in a secondary-care hospital in north-eastern Italy, and to assess the in vitro activity of cefepime-based β-lactam/β-lactamase inhibitor combinations. Methods: We performed a retrospective observational study of consecutive, non-duplicate isolates (September 2024–January 2025). Susceptibility testing was performed using VITEK^® 2 and MIC TestStrip. All isolates underwent single-read Nanopore whole-genome sequencing, with assembly and in silico detection of resistance genes, porin alterations, plasmid replicons and virulence loci. Core-genome single-nucleotide polymorphism analysis with recombination filtering assessed genetic relatedness. Results: Thirteen isolates were recovered from elderly, highly comorbid inpatients, mainly from urine (46.2%) and rectal/fecal screening (38.5%). Twelve patients were classified as colonized, whereas one patient had an infection due to OXA-48-producing K. pneumoniae, with the organism recovered from both blood and urine cultures. All belonged to sequence type 395 and carried blaOXA-48 and blaCTX-M-15, together with a truncated OmpK35 and an OmpK36 loop-3 GD insertion. The yersiniabactin locus ybt16 within integrative conjugative element ICEKp12 was detected in 92.3% of isolates. Core-genome analysis showed high genomic similarity among isolates, consistent with local circulation of an ST395 lineage. Although yersiniabactin may contribute to iron acquisition and bacterial fitness, its presence alone does not establish a hypervirulent phenotype or predict clinical virulence. In the absence of aerobactin, salmochelin, rmpADC, and rmpA2, and without phenotypic virulence testing, these isolates should be regarded as yersiniabactin-positive but non-hypervirulent based on their genomic profile. Core-genome analysis indicated close genetic relatedness among the isolates, consistent with possible local clonal circulation. In a subset, cefepime/enmetazobactam and especially cefepime/zidebactam substantially reduced cefepime minimum inhibitory concentrations. Conclusions: We identified a multidrug-resistant OXA-48-producing K. pneumoniae ST395 lineage circulating locally, predominantly in colonization contexts. Active screening and genomic surveillance may support infection control and inform the potential role of emerging cefepime-based inhibitor combinations.

Chronic Pseudomonas aeruginosa colonisation leads to lung deterioration and poor prognosis in people with cystic fibrosis (pwCF). Early and aggressive therapies can achieve P. aeruginosa eradication, which may recur later. Therefore, determining whether it has resisted therapy or has been newly acquired may guide subsequent treatment(s). This information is crucial also for patients treated with CFTR modulators representing P. aeruginosa after prolonged negativity and to confirm chronic infections. We evaluated the ability of Fourier-transform infrared (FT-IR) spectroscopy to determine intra-patient isoclonality for 103 P. aeruginosa strains isolated from 36 pwCF. Two were chronically and two intermittently colonised; twelve were on modulators with a past P. aeruginosa colonisation; and twenty received eradication therapy, ten of whom were also treated with modulators. FT-IR data were validated by Whole Genome Sequencing (WGS) identification of Sequence Type. FT-IR identified persistence of P. aeruginosa in 24 patients, with a WGS-confirmed positive predictive value of 100% and diagnostic accuracy of 94%. The eradication therapy success rate was 45%, and the time to P. aeruginosa reappearance was similar in both patients with failed eradication treatment and those who initially cleared the infection but later acquired a new strain. Nine patients in modulators showed persistent infections. FT-IR can rapidly determine the clonality of P. aeruginosa isolates, allowing discrimination of recurring versus new infections both in patients with established colonisations and those subjected to eradication therapy representing P. aeruginosa. By overcoming the time-based criteria used to define new infections and by providing the actual success rate of eradication therapies, FT-IR can effectively contribute to the development of efficient therapeutic strategies.

Background: Rapid etiological diagnosis of bacterial meningitis is crucial in children, as delays can lead to neurological sequelae. The BioFire FilmArray Meningitis/Encephalitis (ME) panel is widely used on cerebrospinal fluid (CSF), but its target spectrum may miss healthcare-associated or multidrug-resistant pathogens. We evaluated the diagnostic performance and stewardship-oriented clinical impact of off-label BioFire FilmArray Blood Culture Identification 2 (BCID2) testing on CSF from pediatric patients with suspected bacterial CNS infection and negative ME results. Methods: We retrospectively analyzed CSF samples collected between January 2023 and March 2025 at a tertiary pediatric hospital. In ME-negative cases with persistent suspicion and abnormal CSF parameters, BCID2 was performed off-label on residual CSF aliquots after routine testing, without additional sampling. We assessed pathogen detection, agreement with culture, resistance-gene identification, and documented stewardship actions. Results: Among 76 ME-negative CSF samples tested with BCID2, 23 (30.3%) were positive, all involving organisms not included in the ME panel. BCID2 was concordant with culture in 19/23 cases (82.6%); 4/23 (17.4%) were BCID2-positive/culture-negative, consistent with reduced culture sensitivity in frequently pretreated cases. Resistance genes (VIM, vanA/B, CTX-M) were detected in 30.4% of BCID2-positive samples. Overall agreement with culture was 94.7% (PPA 100%, NPA 93.0%). Escalation was documented in 13/23 episodes (56.5%), discontinuation in 2/23 (8.7%), and confirmation in 9/23 (39.1%), with no de-escalation events; clinical outcomes were not systematically available. Conclusions: In selected ME-negative pediatric cases with abnormal CSF profiles, BCID2 testing on residual CSF provided rapid, clinically meaningful microbiological information that may support antimicrobial optimization.

Next-generation metagenomic sequencing (mNGS) enables the direct and unbiased detection of pathogens from clinical samples, overcoming the limitations of standard methods. It is particularly valuable in immunocompromised patients and in cases of complex infections. We report the case of a man in his 40s, born in North Africa, who was admitted with progressive skin and soft-tissue lesions after a minor foot trauma. The initially localized infection rapidly worsened, leading to bilateral pneumonia, acute respiratory failure, disseminated intravascular coagulation, and death. Histopathological examination revealed granulomatous inflammation with alcohol-resistant bacilli and an undiagnosed cutaneous T-cell lymphoma associated with hemophagocytic syndrome. Conventional microbiological tests identified multiple pathogens, including influenza A virus, herpes simplex virus 1 (HSV-1), Candida albicans , Enterococcus faecalis , Proteus mirabilis , and Pseudomonas aeruginosa ; however, their heterogeneous distribution and isolation from non-sterile sites hindered etiological interpretation. Cultures and molecular assays for Mycobacterium species were negative despite findings of histological examination suggestive of granulomatous inflammation with alcohol-resistant bacilli. To clarify the diagnosis, mNGS was performed on blood, serum, and lymph node samples using host DNA depletion and Illumina sequencing. Bioinformatic analysis revealed a diverse microbial landscape, with the detection of Fusarium pseudograminearum , Mycobacterium canettii, and Ralstonia sp., alongside low-level viral sequences [Epstein–Barr virus ( EBV ) and HSV-1 ]. These results reflected the patient’s severe immune deficiency, characterized by a marked depletion of CD8 ⁺ T cells and NK cells. Although the results became available too late to influence treatment, mNGS provided crucial diagnostic insights, demonstrating its ability to uncover hidden or rare pathogens. Early application of mNGS could significantly improve diagnostic precision and therapeutic decisions in critically ill immunocompromised patients.

Background: Since 2022, invasive Group A Streptococcus (GAS) infections have increased, mainly due to the spread of specific emm-types, such as emm1. As therapy may depend on the emm-type, rapid and cost-effective identification is crucial. Fourier-transform infrared spectroscopy (FTIR) has emerged as a promising alternative to sequencing for GAS typing. We applied machine learning (ML) to FTIR spectra to build a predictive model for emm-type identification. Methods: Twenty-four GAS strains were analyzed by whole-genome sequencing and FTIR. The model was trained on twenty-one strains (emm-types: 1, 3, 4, and 6), using leave-one-out cross validation (LOOCV). To test the model’s ability to avoid false positive results, the model was also tested with three strains belonging to emm-types not included in the training of the model (emm-types: 12, 89, and 75). Results: An artificial neural network trained for 400 cycles achieved the highest accuracy (90.7%) out of the thirteen different models tested. When the three strains belonging to emm-types not included in the model were predicted with this model, it produced low score values, confirming its ability to avoid false positive results. Conclusions: We developed a preliminary and proof-of-concept model capable of accurately predicting the four most-prevalent emm-types in our setting, including the highly virulent emm1. These findings support FTIR combined with ML as a rapid, low-cost tool for GAS typing, with potential for real-time clinical applications to guide timely treatment decisions. However, as a proof-of-concept study, the relatively small sample size and limited emm-type diversity underline the need for further validation with larger and more diverse datasets.

Candida viswanathii has been sporadically reported in Asia and South America but not in Europe. This study reports the first European outbreak of C. viswanathii in a paediatric hospital, outlining diagnostic challenges and containment measures. Fifteen C. viswanathii isolates were recovered from blood cultures of consecutive pediatric patients admitted to intensive care units between April and August 2025. Identification was performed using MALDI-TOF MS, chromogenic media, Fourier-transform infrared (FT-IR) spectroscopy, and whole-genome sequencing (WGS). Antifungal susceptibility testing was performed by broth microdilution. All isolates were initially misidentified as C. tropicalis by MALDI-TOF MS and undetected by the FilmArray BCID2 panel. WGS confirmed C. viswanathii, and FT-IR analysis revealed clonally related strains, indicating an outbreak. Colonies displayed a distinct deep-blue color on chromogenic CHROMagar™ medium. Elevated fluconazole minimum inhibitory concentrations were observed, while isolates remained susceptible to echinocandins and amphotericin B. A multidisciplinary infection-control response halted transmission within four weeks. This investigation documents the first C. viswanathii outbreak in Europe, highlighting diagnostic limitations of current commercial tools and the need for updated databases. Integration of FT-IR spectroscopy and WGS facilitated outbreak detection and containment, underscoring the importance of advanced diagnostics and surveillance for emerging fungal pathogens.

Human adenoviruses (HAdV) comprise more than 100 genotypes with species-specific differences in tropism and immune response and can cause severe infections in immunocompromised patients. This study aimed to characterise the HAdV species involved in pediatric infections to assess their clinical impact and guide future therapeutic strategies based on AdV-specific T-cell responses. Between January and October 2024, 595 pediatric HAdV diagnoses were made at the Bambino Gesù Children’s Hospital (Rome), and whole-genome sequencing was performed on 60 samples. Most patients (91.7%) were hospitalised, including both immunocompetent (75%) and immunocompromised (25%) children. Gastrointestinal and respiratory symptoms were more common in immunocompetent patients, whereas immunocompromised patients experienced longer hospitalisations and persistent viral infections. Species F (F41) was most prevalent (63.3%), especially among immunocompetent patients, while species C and A predominated in immunocompromised children, with species A associated with severe disease. Viral loads were significantly higher for species F than for species A and C, independent of immune status. Co-infections were frequent (63.3%), with species C particularly linked to them. In conclusion, HAdV distribution differed by immune status, with species F predominating in immunocompetent children and species C and A more common in immunocompromised patients. Whole-genome sequencing may enhance surveillance, enable earlier diagnosis, and support the development of genotype-specific immunotherapies.

Background/Objectives: Paediatric osteoarticular infections (POAIs) present unique diagnostic and therapeutic challenges. Microbiological culture (MC) is typically time-consuming and lacks sensitivity, especially when patients have received antibiotics. The BIOFIRE^® Joint Infection Panel (BJIP) is a syndromic molecular assay for the direct identification of most pathogens causing POAIs. Methods: We evaluated BJIP in 17 synovial fluids, and then, we retrospectively assessed its utility in 93 off-label specimens (i.e., 25 purulent fluids/biopsies and 68 whole blood samples). All specimens were collected from October 2022 to March 2024 from paediatric patients admitted at the Bambino Gesù Children’s Hospital in Rome. Results: A bacterial pathogen was isolated in only one of 17 synovial fluid cultures, while BJIP identified eight additional microorganisms in MC-negative cases. The most frequently detected pathogen was S. aureus (44.5%, 4/9). BJIP performance in synovial fluids showed an overall positive percentage agreement (PPA) and negative percentage agreement (NPA) of 100% and 88.1%, respectively, compared to MC. All positive results (n/N = 9/17) were considered medically significant, with an increase in NPA to 100%. In purulent fluids/biopsies, BJIP and MC were concordant in 72% of cases (n/N = 18/25), with a per-sample PPA and NPA of 90% and 60%, respectively. For whole blood samples, almost all samples were negative by both methods (i.e., reference blood culture and BJIP), and the molecular test did not enable any further microbiological diagnosis. Conclusions: The BIOFIRE^® Joint Infection Panel rapidly and accurately enabled or excluded a diagnosis of a POAI (~1 vs. 24–96 h for MC), optimising antimicrobial therapy.

Background: Bloodstream infections affect up to 20% of pediatric cancer patients receiving intensive care, contributing significantly to morbidity and mortality, with infection-related mortality rates reported to be as high as 16%. Methods: The identification of microorganisms directly from whole blood is difficult due to several factors, such as interference from host genomic material, low bacterial load, the endogenous components of whole blood and exogenous substances, which can interfere with the identification process. Nevertheless, rapid microbial diagnosis remains of paramount importance in these patients. Results and Conclusion: Here, we present the first case of bacterial pathogen identification directly from whole blood using MALDI-TOF mass spectrometry in an onco-hematological pediatric patient affected by sepsis and admitted to Bambino Gesù Children’s Hospital (IRCCS) in Rome, Italy.

Background: Infant meningitis, particularly caused by Escherichia coli, remains a life-threatening condition, especially in premature and low-weight infants. Infections of the central nervous system can be fatal, necessitating prompt diagnosis and appropriate treatment. Acute infections caused by various pathogens, including E. coli, often present with similar clinical symptoms. The rapid identification of pathogens and their antimicrobial resistance mechanisms is critical for timely and effective treatment. We report the case of an 8-month-old patient who presented with fever, diarrhea, and convulsive seizures and was subsequently diagnosed with meningitis. Despite initial empirical treatment with ceftriaxone, the patient’s condition worsened. Methods: At Bambino Gesù Children’s Hospital, molecular diagnostic tools, including the FilmArray Meningitis/Encephalitis and Blood Culture Identification panels, were employed. Results: Although the Meningitis panel did not detect any pathogens due to the lack of the specific bacterial target, the off-label use of the Blood Culture Identification panel identified a non-K1 Escherichia coli strain carrying the CTX-M resistance gene, an extended-spectrum beta-lactamase (ESBL). Despite the rapid diagnostic approach and adjustment of antibiotic therapy, the patient succumbed to the infection due to the strain’s high virulence and multidrug resistance. Whole-genome sequencing further characterized the strain, revealing that it belonged to the ST131 group, a highly resistant and virulent strain associated with sepsis. Conclusions: This case highlights the importance of integrating advanced molecular diagnostics, such as whole-genome sequencing, with traditional methods to improve pathogen detection, especially in cases of emerging resistant strains that are not covered by standard diagnostic panels. It also emphasizes the need for the continuous adaptation of diagnostic tools to include non-K1 E. coli strains for more comprehensive and timely meningitis diagnosis.