The rapid and accurate detection of SARS-CoV-2 in environmental settings is crucial for effective public health management during the COVID-19 pandemic. This study compares the performance of the Reverse Transcription quantitative polymerase chain reaction (RT-qPCR) and the Reverse Transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 detection from 100 surface samples collected in healthcare environments. The reference method, RT-qPCR, identified a percentage of 25% of positive samples, while RT-LAMP detected a percentage of 27% of positive surfaces. Our findings reveal a sensitivity of 32% and specificity of 75% for RT-LAMP, with a positive predictive value of 30% and a negative predictive value of 77%. The overall accuracy and concordance with RT-qPCR was 64% for both methods. Despite its lower sensitivity compared to RT-qPCR, RT-LAMP had an advantage due to its rapid screening and environmental surveillance, which is particularly useful for confirming negative results. These results underscore the potential of RT-LAMP not only as a valuable method in the environmental monitoring of SARS-CoV-2 but also as a system to control the sanitation process in ordinary and emergency conditions, providing further optimization and validation for its reliability in routine surveillance and outbreak response efforts.

Contamination of environmental surfaces by nosocomial pathogens like Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus), and Enterococcus spp. poses significant health risks worldwide. However, gold-standard detection methods are too time-consuming and labor-intensive. This study aimed to optimize loop-mediated isothermal amplification (LAMP) as a rapid, innovative, and cost-effective approach, comparing its effectiveness with the gold-standard cultural method. Sterile surfaces (24 cm²) were contaminated in duplicate with different concentrations of P. aeruginosa, S. aureus, and Enterococcus faecalis (E. faecalis) reference stains. For each pair of contaminated surfaces, one was analyzed using the agar contact plate method (UNI EN 17141:2021), while the other was analyzed using LAMP, following three different pre-incubation times (three, six, and nine hours). The sensitivity and accuracy of LAMP for P. aeruginosa improved with longer incubation times, reaching a value of 1.00 at nine hours, while the specificity and positive predictive value (PPV) remained at 1.00 regardless of the incubation time. For S. aureus, LAMP achieved a sensitivity, specificity, accuracy, PPV, and negative predictive value (NPV) of 1.00 across all incubation times. Finally, for E. faecalis, sensitivity increased from 0.57 at three hours to 1.00 at six and nine hours, with a high specificity, accuracy, PPV, and NPV from six hours onwards. These findings showed that LAMP can be used as a rapid and reliable alternative to gold-standard methods for detecting pathogens on surfaces. The high sensitivity and specificity achieved, especially at six and nine hours of pre-incubation, suggested its use for real-time monitoring in healthcare settings. Further research in real-world environments is needed to confirm these findings.

Hospital environments are recognized as significant reservoirs of nosocomial pathogens, contributing to the onset of healthcare-associated infections (HAIs). Timely microbiological monitoring is essential to mitigate infection risks. However, gold-standard methods based on culture and biochemical techniques are time-consuming and may underestimate microbial contamination, potentially delaying interventions. This study proposes a novel approach for surface monitoring using loop-mediated isothermal amplification (LAMP) for the rapid detection of key nosocomial pathogens, such as Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus spp. A total of 145 surface samples were collected from six Italian hospitals and analyzed by both standard culture and LAMP methods, following two different incubation times (6 and 9 h) using pre-enrichment medium. Comparison with the reference method revealed that the LAMP assay achieved a sensitivity of 1.00 for all target pathogens at both 6 and 9 h of incubation. Specificity values were slightly higher at 6 h compared to 9 h: 0.93 vs 0.90 for P. aeruginosa, 0.91 vs 0.89 for Enterococcus spp., while remaining 0.92 for S. aureus, at both incubation times. These results suggest that a 6-h incubation period offers an optimal balance between speed and diagnostic accuracy, making LAMP a promising tool for rapid microbiological surveillance in healthcare settings.

This study presents the characterization of a novel Legionella species isolated in Italy over three different years from one company and two hospitals. Starting from standard techniques such as culture of water samples, agglutination test, MALDI-TOF MS and gene sequencing analysis used to identify the isolates, genomic and metabarcoding approaches were subsequently employees to further characterize the species. Legionella contamination ranged from 400 to 700 CFU/L. The tiny colonies displayed atypical morphology compared to typical Legionella features, although they grew on BCYE medium supplemented with L-cysteine. Microscopic and phenotypic analyses revealed Gram-stain negative, Ziehl-Neelsen-negative, rod-shaped, motile cells capable of growing at 32-37 °C, including on selective media such as GVPC and MWY. The isolates tested positive for oxidase and gelatinase activity. Fatty acid profiling identified the dominant components as Summed Features 3 (C16:1 ω7c/C16:1 ω6c, 28.9%), C16:0 iso (18.4%), and C15:0 anteiso (15.4%). Ubiquinone Q13 was the major quinone. Sequence analysis of the mip and rpoB genes showed 98.2% and 95.1% similarity, respectively, to L. feeleii (WO-44C ATCC 35072^T). Whole genome sequencing (WGS) revealed a GC content of 41.5%, a dDDH value of ≤54.9%, and an ANI of 94.06% with L. feeleii (WO-44C ATCC 35072^T), supporting the classification of a novel species within the genus Legionella. Furthermore, taxonomic resolution of water samples revealed the presence of 168 bacterial genera, including several respiratory, opportunistic, and zoonotic pathogens, as well as seven Legionella species. The name Legionella petroniana sp. nov. is proposed, with strain 31fI33^T (=DSM 114357^T=CCUG 76442^T) designated as type strain.

Nosocomial pathogens persist on hospital surfaces contributing to healthcare-associated infections (HAIs), especially among vulnerable patients and in the presence of multidrug-resistant strains. Environmental surveillance is essential to prevent cross-contamination and support timely infection control interventions. However, conventional culture-based methods, although considered the gold standard, are labor-intensive and time-consuming, often delaying critical responses. This study evaluated loop-mediated isothermal amplification (LAMP) as a rapid screening tool for hospital environmental monitoring. A total of 100 surface samples were collected from different hospital wards and analyzed using both culture and LAMP assays targeting six major HAI-related pathogens: Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus spp., Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii. LAMP showed excellent performance, with sensitivity of 1.00 for all targets and specificity ranging from 0.94 to 1.00. No statistically significant differences were observed between LAMP and culture results (p > 0.05). LAMP may represent a valuable complementary method for routine environmental surveillance.

Airborne microbial contamination and particulate matter in operating rooms (ORs) contribute to the risk of surgical site infections (SSIs). However, few studies have integrated culture‐based approaches with MALDI‐TOF MS identification and metagenomic NGS during real surgical activity. This study combines microbiological, molecular, particulate, and procedural data to provide a multifactorial view of intraoperative contamination dynamics. This study aimed at characterizing airborne microbiota, particulate matter, and key procedural determinants of contamination in ORs. Forty‐two surgical procedures across three specialties were monitored. Airborne microorganisms were isolated using culture‐based methods and identified with the MALDI Biotyper, whereas the OR airbiome was assessed using shotgun metagenomic sequencing. Airborne particle concentrations (≥ 0.5 and ≥5 μ m), microclimatic parameters, staff presence, door openings, electrosurgical use, and procedural order were recorded. Statistical analyses included Wilcoxon and Kruskal–Wallis tests with Dunn′s post hoc comparisons, Kendall′s τ and Spearman′s ρ correlations, and rank‐based effect sizes. Airborne microbial contamination was low in both ISO 5 and ISO 7 rooms (mean 14.02 ± 6.88 CFU/m ³ ), with a predominance of skin‐associated taxa. Culture‐based methods detected viable, fast‐growing species, whereas NGS revealed additional environmental, slow‐growing, and anaerobic taxa, including clinically significant bacteria and yeasts such as Cutibacterium acnes ( C . acnes ), Finegoldia magna ( F . magna ), and Malassezia restricta ( M. restricta ). Cross‐method concordance was weak to moderate (Spearman ^′ s ρ = 0.35; Kendall ^′ s τ = 0.24), indicating complementary detection. Median particle concentrations were significantly higher in ISO 7 than in ISO 5 ORs. In ISO 7 rooms, particle loads varied by specialty and tended to be higher during open procedures. Integrated analyses showed a decoupling of microbial and particulate contamination in ISO 5 rooms, whereas ISO 7 rooms exhibited more interconnected contamination dynamics, with significant associations between electrosurgical device use and airborne microbial contamination ( ρ = 0.45, p = 0.025), between fine (≥ 0.5 μ m) and coarse (≥ 5 μ m) particle concentrations ( ρ = 0.65, p < 0.001), and an inverse association between procedure order and staff count ( ρ = −0.66, p < 0.001). Integrating culture‐based and molecular techniques may provide a more comprehensive characterization of the airborne microbiota in ORs. The findings suggest that ISO 5 laminar‐flow systems are associated with more stable airborne contamination patterns, whereas ISO 7 rooms exhibit greater interactions between airborne contamination and procedural variables. Incorporating molecular profiling and particle metrics into OR surveillance may support environmental monitoring strategies relevant to infection prevention and control.