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.

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.

Evaluation of 16S-Based Metagenomic NGS as Diagnostic Tool in Different Types of Culture-Negative Infections.

Bacterial infections pose significant global health challenges, often underestimated due to difficulties in accurate diagnosis, especially when culture-based diagnostics fail. This study assesses the effectiveness of 16S-based metagenomic next generation sequencing (NGS) for identifying pathogens in culture-negative clinical samples across various medical settings. Overall, 48% of samples were collected from orthopedics, 15% from neurosurgery, and 12% in cardiac surgery, among others. The detection rate of monomicrobial infections was 68.6%, and 5.7% for polymicrobial infections. In addition, NGS detected bacteria in all samples from the lungs, head and neck, and eye specimens. Cutibacterium acnes (11%, 12/105) was the most frequent microorganism, followed by Staphylococcus epidermidis (10.4%, 11/105), and Staphylococcus aureus (9.5%, 10/105). In conclusion, 16S-targeted metagenomic sequencing enhances pathogen detection capabilities, particularly in instances where traditional cultures fail. By the combination of NGS and bacterial cultures, microbiologists might provide a more accurate diagnosis, guiding more effective treatments and potentially reducing healthcare costs associated with empirical treatments.

Traditional Cultures versus Next Generation Sequencing for Suspected Orthopedic Infection: Experience Gained from a Reference Centre.

(Background) The diagnosis and the antimicrobial treatment of orthopedic infection are challenging, especially in cases with culture-negative results. New molecular methods, such as next-generation sequencing (NGS), promise to overcome some limitations of the standard culture, such as the detection of difficult-to-grow bacteria. However, data are scarce regarding the impact of molecular techniques in real-life scenarios. (Methods) We included cases of suspected orthopedic infection treated with surgery from May 2021 to September 2023. We combined traditional cultures with NGS. For NGS, we performed a metagenomic analysis of ribosomal 16s, and we queried dedicated taxonomic libraries to identify the species. To avoid false positive results, we set a cut-off of 1000 counts of the percentage of frequency of reads. (Results) We included 49 patients in our study. Our results show the presence of bacteria in 36/49 (73%) and 29/49 (59%) cases studied with NGS and traditional cultures, respectively. The concordance rate was 61%. Among the 19/49 discordant cases, in 11/19 cases, cultures were negative and NGS positive; in 4/19, cultures were positive and NGS negative; and in the remaining 4/19, different species were detected by traditional cultures and NGS. (Conclusions) Difficult-to-grow microorganisms, such as slow-growing anaerobic bacteria, were better detected by NGS compared to traditional culture in our study. However, more data to distinguish between true pathogens and contaminants are needed. NGS can be an additional tool to be used for the diagnosis of orthopedic infections and the choice of appropriate antimicrobial therapy.

Presence and infectivity of SARS-CoV-2 virus in wastewaters and rivers.

The presence of SARS-CoV-2 in raw wastewaters has been demonstrated in many countries affected by this pandemic. Nevertheless, virus presence and infectivity in treated wastewaters, but also in the receiving water bodies are still poorly investigated. In this study, raw and treated samples from three wastewater treatment plants, and three river samples within the Milano Metropolitan Area, Italy, were surveyed for SARS-CoV-2 RNA detection by means of real time RT-PCR and infectivity test on culture cells. SARS-CoV-2 RNA was detected in raw, but not in treated wastewaters (four and two samples, respectively, sampled in two dates). The isolated virus genome was sequenced, and belonged to the strain most spread in Europe and similar to another found in the same region. RNA presence in raw wastewater samples decreased after eight days, probably following the epidemiological trend estimated for the area. Virus infectivity was always null, indicating the natural decay of viral pathogenicity in time from emission. Samples from receiving rivers (three sites, sampled in the same dates as wastewaters) showed in some cases a positivity to real time RT-PCR, probably due to non-treated, or inefficiently treated discharges, or to the combined sewage overflows. Nevertheless, also for rivers infectivity was null. Risks for public health should be limited, although a precautionary approach to risk assessment is here advocated, giving the preliminary nature of the presented data.