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Abstract Background Sepsis represents a time-sensitive disease requiring early therapeutical intervention to avoid adverse patient outcomes. Rapid microbiological diagnosis is essential to investigate sepsis aetiological agents. The FAST™ system (Qvella, ON, Canada) provides a concentrated microbial suspension, known as a Liquid Colony™ (LC), directly from positive blood samples (PBCs) in 30–40 min to perform rapid identification (ID) and antimicrobial susceptibility testing (AST). Methods Qvella’s FAST™ System and FAST PBC Prep cartridges were tested on PBCs from the Policlinico Hospital of Catania during a six-month study. Two millilitres of PBC were converted into an LC for rapid ID and AST using Bruker Biotyper Sirius MALDI and BD Phoenix systems. Standard of care (SOC) methods were used as a reference, requiring 48–72 h. Agreement between the innovative technology and the standard method was calculated. Results FAST System processing was performed on 100 monomicrobial PBCs. Median turnaround times from blood cultures flagging positive to ID and AST completion were 2 and 26 h respectively. Therefore, the LC procedure was 24 h faster than the median turnaround times for SOC methods. 100% ID identification concordance was observed across 48 Gram-negative bacteria, 42 Gram-positive bacteria and 11 yeast for the genus level. 78% of Gram-negative and 95% of Gram-positive bacteria were resistant to ≥ 2 antimicrobial agents, including 45% (15/33) carbapenem-resistant enteric Gram-negative bacteria and 90% (28/31) oxacillin-resistant staphylococci. An AST essential agreement of 100% was observed due to the absence of MIC discrepancies > 1-fold dilution. Categorical errors were not observed due to the absence of MIC categorization discordances. Yeast AST was not performed. Conclusions The Qvella FAST System produces an LC that reliably reflects the MALDI spectra and phenotypic antimicrobial susceptibility profile of microbial cells growing in the blood culture. Timely processing of PBCs with the Qvella FAST System enables sepsis diagnostic confirmation 1 day sooner than the standard methods. In line with these results, it is vital now to focus attention on establishing best practices for incorporating this strategic tool into the clinical microbiology laboratory workflow.

Digital polymerase chain reaction (dPCR) has emerged as a groundbreaking technology in molecular biology and diagnostics, offering exceptional precision and sensitivity in nucleic acid detection and quantification. This review highlights the core principles and transformative potential of dPCR, particularly in infectious disease diagnostics and environmental surveillance. Emphasizing its evolution from traditional PCR, dPCR provides accurate absolute quantification of target nucleic acids through advanced partitioning techniques. The review addresses the significant impact of dPCR in sepsis diagnosis and management, showcasing its superior sensitivity and specificity in early pathogen detection and identification of drug-resistant genes. Despite its advantages, challenges such as optimization of experimental conditions, standardization of data analysis workflows, and high costs are discussed. Furthermore, we compare various commercially available dPCR platforms, detailing their features and applications in clinical and research settings. Additionally, the review explores dPCR's role in water microbiology, particularly in wastewater surveillance and monitoring of waterborne pathogens, underscoring its importance in public health protection. In conclusion, future prospects of dPCR, including methodological optimization, integration with innovative technologies, and expansion into new sectors like metagenomics, are explored. [ABSTRACT FROM AUTHOR]

Objectives: Carbapenem-resistance is a challenging healthcare concern and require specific stewardship programs. Monitoring workflows include the identification from surveillance samples, such as rectal swabs. Although culture assays represent the gold standard, data report a significant effectiveness in detecting carbapenemases genes directly from rectal swabs. The aim of this study was to evaluate the REALQUALITY Carba-Screen kit (AB ANALITICA, Padova, Italy) in detecting carbapenemases genes directly from rectal swabs, also comparing its effectiveness to culture assays results. A next-generation sequencing (NGS) was performed to investigate the positive samples about resistance markers and sequence type (ST). Methods: A number of 136 rectal swabs were collected from the University Hospital Policlinico of Catania critical wards. The samples simultaneously underwent culture and molecular assays (REALQUALITY Carba-Screen kit). The molecular method included two-steps. The first step (1 h and 6 min) rapidly excluded negative samples, while the second one (1 h and 6 min) included only positive samples for a resistance confirmation. All the positive culture samples underwent NGS analysis. Results: Statistical evaluations demonstrated high sensitivity (100%) and detection rates (92.6%) for the REALQUALITY Carba-Screen kit, which mostly correlated to the standard workflow. All the culture positive results matched the positive molecular results, which were mainly confirmed by the NGS resistome analysis. The identified ST appeared to be diversified and different from the clinically significative strains of the same setting, furnishing interesting epidemiological evidence. Conclusion: The molecular detection allowed a coordinate approach in a high-prevalence multi-drug-resistance area. The rapid identification with a multi-step procedure accelerated the infection control procedures, while the preliminary negative results reduced the overtreatment episodes. The molecular method efficacy was confirmed through the NGS. In conclusion, the molecular screening could initially lead to a more conservative approach, which may be reevaluated after a culture result about the microorganisms' identification and susceptibility profile. [ABSTRACT FROM AUTHOR]

Development of in vivo daptomycin resistance (DAP-R) among Staphylococcus aureus clinical isolates, in association with clinical treatment failures, has become a major therapeutic problem. This issue is especially relevant to methicillin-resistant S. aureus (MRSA) strains in the context of invasive endovascular infections. In the current study, we used three well-characterized and clinically-derived DAP-susceptible (DAP-S) vs. resistant (DAP-R) MRSA strain-pairs to elucidate potential genotypic mechanisms of the DAP-R phenotype. In comparison to the DAP-S parental strains, DAP-R isolates demonstrated (i) altered expression of two key determinants of net positive surface charge, either during exponential or stationary growth phases (i.e., dysregulation of dltA and mprF), (ii) a significant increase in the D-alanylated wall teichoic acid (WTA) content in DAP-R strains, reflecting DltA gain-in-function; (iii) heightened elaboration of lysinylated-phosphatidylglyderol (L-PG) in DAP-R strains, reflecting MprF gain-in-function; (iv) increased cell membrane (CM) fluidity, and (v) significantly reduced susceptibility to prototypic cationic host defense peptides of platelet and leukocyte origins. In the tested DAP-R strains, genes conferring positive surface charge were dysregulated, and their functionality altered. However, there were no correlations between relative surface positive charge or cell wall thickness and the observed DAP-R phenotype. Thus, charge repulsion mechanisms via altered surface charge may not be sufficient to explain the DAP-R outcome. Instead, changes in the compositional or biophysical order of the DAP CM target of such DAP-R strains (i.e., increased fluidity) may be essential to this phenotype. Taken together, DAP-R in S. aureus appears to involve multi-factorial and strain-specific adaptive mechanisms.

The MIC value definition faithfully reflects antimicrobial sensitivity, profoundly impacting the infection's clinical outcome. Our study aimed to evaluate the Accelerate PhenoTM System in defining the importance of fast phenotypic susceptibility data. A number of 270 monomicrobial samples simultaneously underwent standard procedures and fast protocols after a contemporary Gram stain. Finally, we provided Turn-around Time (TAT) and statistical evaluations. The fast technology required a medium value of 7 h to complete ID and AST profiles. Although there were some spectrum limitations, it revealed an optimal success rate in microbial identification directly from positive blood cultures. The Gram-negative AST reached a 98.9% agreement between the Accelerate Pheno™ System and the standard method. In addition, the Gram-positive AST gathered a 98.7% agreement comparing the same systems. The chance to rapidly provide precise MIC values is one of the last frontiers in clinical microbiology, especially in high-prevalence antimicrobial resistance areas. [ABSTRACT FROM AUTHOR]