Background/Objectives: Bloodstream infections (BSIs) are a global healthcare issue associated with high mortality rates. Rapid diagnosis is of importance for the early selection of targeted therapy to improve patient outcomes. The use of rapid molecular assays with positive blood culture (BC) allows the identification (ID) of pathogens and the most relevant resistance determinants (RDs) in a shorter turnaround time, compared to standard culture. In this study, the performances of a new syndromic panel to determine the IDs and RDs of Gram-negative (GN) and Gram-positive (GP) bacteria were investigated in comparison with a standard-of-care (SoC) workflow. Methods: Two hospitals processed residual positive BC samples from non-replicated patients using Molecular Mouse (MM) Sepsis panels (Alifax, Padova, Italy) for GP ID, GN ID and RD detection. Results were compared with an SOC workflow based on subculture, ID by MALDI-ToF mass spectrometry, phenotypic antibiogram, and real-time PCRs for RDs from isolated colonies. Results: A total of 140 and 136 residual positive BC samples were found to be valid for MM-ID and RD, respectively, yielding 76 GN and 76 GP species. Overall ID agreement at the species level was 136/152 (89%). RD agreement was 144/146 (99%). Regarding GN and GP species, ID agreement was 68/76 (89%) and 70/76 (92%), respectively. Conclusions: MM showed high sensitivity in RD detection; however, some discrepancies with results of the SoC workflow were observed, represented by reduced sensitivity for some species-specific IDs. Panel size and compact instrument dimension can be seen as the principal advantage of this modular molecular assay for the rapid detection of pathogens responsible for BSIs.

Babesiosis, a tick-borne disease caused by protozoa of the genus Babesia, is endemic in North America, but rarely reported in Europe. We report a case involving a splenectomised 72-year-old man from New Hampshire, USA, who was admitted at Careggi University Hospital (Florence, Italy), complaining of a febrile syndrome appeared during a trip to central Italy. After the laboratory flagged the presence of intra-erythrocytic ring-shaped elements, malaria was initially suspected but subsequently excluded by molecular testing. Based on epidemiological and clinical history, Babesia microti infection was suspected and confirmed by molecular test on peripheral blood. Oral atovaquone and intravenous azithromycin were administered for 7 days, switched to oral therapy following clinical improvement and parasitemia clearance. In non-endemic regions, the typical clinical features of this case might not raise suspicion. Nevertheless, rapid diagnosis and treatment are key to ensure favorable clinical outcomes, since babesiosis can cause rapidly aggravating illness, particularly in splenectomised patients.

Neurorehabilitation units (NRUs) are long-term care settings associated with multiple risk factors for carriage and infection by multidrug-resistant organisms (MDROs), including prolonged hospitalization, the use of invasive medical devices, high antibiotic exposure, and multiple comorbidities. We report the case of a patient admitted to an NRU for rehabilitation after cerebral hemorrhage who developed complex co-colonization by several MDROs, including Pseudomonas aeruginosa producing either FIM or VIM metallo-β-lactamases, Acinetobacter baumannii producing OXA-23 serine-carbapenemase, Klebsiella pneumoniae producing either KPC or OXA-48 serine-carbapenemases or both, Proteus mirabilis producing VEB extended-spectrum β-lactamase, and vancomycin-resistant Enterococcus faecium of the VanA type. During the 22-month hospitalization, the patient experienced several infection episodes, some of which were apparently related with colonizing MDROs. This case highlights that co-colonization by multiple MDROs may occur in patients admitted to NRUs in endemic settings, and emphasizes the importance of implementing targeted infection prevention and control strategies in these healthcare environments.

Objectives Oxazolidinones are antibiotics of remarkable clinical importance, and resistance to these drugs is a matter of concern. This study investigated the presence of optrA, poxtA and cfr transferable oxazolidinone resistance determinants among enterococci and other non-obligate anaerobic fecal Gram-positives from healthy children living in a rural area of Bolivia. Methods Fecal samples were collected in transport medium and screened for isolates growing on Colistin-Nalidixic Acid blood agar containing florfenicol 16 mg/L. Isolates were identified by MALDI-ToF mass spectrometry and subjected to Real-time PCR to detect the presence of optrA, poxtA and cfr genes. Antimicrobial susceptibility to florfenicol, vancomycin and linezolid was tested by reference broth microdilution. Results Overall, 184/420 (43.8%) faecal samples yielded growth on the selective medium and 241 isolates of 13 different species of Lactobacillales (mostly Enterococcus spp. but also lactococci and Vagococcus teuberi) were obtained for further investigation. Most of them carried either optrA (182/241) or poxtA (43/241) or a combination thereof (7/241). cfr was detected in 5/241 isolates, always in combination with the other genes. Conclusions Present findings report the highest prevalence of faecal carriage of optrA- and poxtA-positive commensals so far observed in healthy subjects, raising concerns about the potential clinical and epidemiological implications.

Background: Stenotrophomonas maltophilia infections represent a clinical challenge in treating frail and immunocompromised patients. Alternatives to trimethoprim-sulfamethoxazole (SXT) are needed, with cefiderocol (FDC) representing a promising option, but clinical evidence is limited; moreover, data to support the superiority of mono or combination therapy are lacking. Case presentation: We describe the case of a 55-year-old female patient with a tunneled hemodialysis central venous catheter (HD-CVC)-associated bloodstream infection caused by S. maltophilia that, after failure of a prolonged SXT monotherapy, was successfully treated by HD-CVC replacement followed by intravenous cefiderocol (FDC) and levofloxacin (LVX). Conclusions: FDC represents an interesting option for complex cases of S. maltophilia bloodstream infections, and the combination with LVX might add benefit in cases associated with biofilm formation on intravascular devices.

The accuracy and turnaround time (TAT) of the QuickMIC system (QMIC) for rapid antimicrobial susceptibility testing (AST) of Gram-negative pathogens from positive blood cultures were evaluated, in comparison with a standard-of-care workflow based on culture and AST with broth microdilution. Essential agreement (EA) and bias were evaluated according to the ISO 20776-2:2021, category agreement (CA), categorical overestimation (CO), and underestimation (CU) rates were evaluated according to the European Committee on Antimicrobial Susceptibility Testing(EUCAST) breakpoints, and major discrepancy (MD) and very major discrepancy (VMD) rates were evaluated according to the Food and Drug Administration (FDA) guidelines and breakpoints. QMIC yielded evaluable data for 103/118 blood cultures (87.3%), including Enterobacterales (N = 82), Pseudomonas aeruginosa (N = 16), and Acinetobacter baumannii complex (N = 5). Overall, 41 isolates (39.8%) were positive for extended-spectrum β-lactamases and/or carbapenemases. QMIC exhibited EA and CA values of 89.5% and 93.3%, respectively, with values <90% were reported for cefepime, meropenem, and piperacillin-tazobactam. Mean bias was -23.1%, with values >±30% observed with colistin and piperacillin-tazobactam. Overall, CO and CU were observed in 2.3% and 4.4% of cases. MD and VMD with FDA breakpoints were observed in 1.5% and 8.9% of cases, respectively. The lowest accuracy of QMIC in terms of CU was observed with cefepime, colistin, and gentamicin and in terms of VMD with ceftazidime-avibactam, meropenem, and piperacillin-tazobactam. The mean TAT for QMIC was 192 ± 18 min. QMIC demonstrated the potential for rapid AST, with a mean time to result of less than 4 hours, and an overall acceptable accuracy, except with some β-lactams. Further studies are warranted to assess the potential impact of QMIC implementation on clinical outcomes.IMPORTANCEThe introduction of rapid antimicrobial susceptibility testing (AST) systems for positive blood cultures (BCs) is one of the most promising technological advancements to improve bloodstream infections diagnosis, by providing faster results useful for antimicrobial stewardship. The value of such systems is expected to be higher in settings of high endemicity of antimicrobial resistance, where phenotype prediction is more difficult, and rates of inaccurate empirical therapies are expected to be higher. In this study, we investigated the accuracy and turnaround time (TAT) of a new rapid AST system for positive BCs, in comparison with a standard-of-care workflow based on broth microdilution, including several cases positive for extended-spectrum β-lactamase- and/or carbapenemase-producing Gram negatives. TAT was 192 ± 18 min, and a good accuracy was observed for testing some agents (amikacin, gentamicin, ciprofloxacin, colistin, and ceftazidime-avibactam) but a lower accuracy for testing other agents (cefepime, meropenem, and piperacillin-tazobactam). Further assessment of this technology, after improving accuracy, is warranted to evaluate the clinical impact.

NDM-type metallo-β-lactamases (MBLs) are among the most widespread acquired carbapenemases in carbapenem-resistant Enterobacterales. As with other β-lactamases, allelic variability occurs among NDM-type MBLs, with almost 100 variants so far reported, differing by single or multiple amino acid substitutions or insertions, which may have implications for enzymatic activity. In this study, we report on a novel NDM variant, NDM-63, identified in a carbapenem-resistant ST147 Klebsiella pneumoniae from a surveillance rectal swab. Compared to NDM-1, NDM-63 features an original array of changes in the L3 loop, including deletion of phenylalanine at position 70 and two amino acid substitutions (G69S and A72H), due to a four-nucleotide deletion plus a nucleotide insertion in the gene region encoding the L3 loop. When expressed in Escherichia coli under isogenic conditions, NDM-63 conferred a resistance profile overall similar to NDM-1, but exhibiting a lower level of resistance to carbapenems and cefepime, while remaining susceptible to inhibition by taniborbactam. Present findings expand current knowledge on the structural plasticity of NDM-type MBLs and highlight that variability in the L3 loop, which contributes to delimitation of the active site, could also tolerate amino acid deletions without loss of enzymatic activity. A virtually identical K. pneumoniae carrying a non-functional blaNDM allele entailing only the nucleotide insertion observed in blaNDM-63 (which might have played a role in the evolution of blaNDM) was also isolated from a bloodstream infection that occurred in the same patient, yielding a misleading result of molecular diagnostic testing due to the lack of enzyme activity despite the presence of the target gene.

During the COVID-19 pandemic, some studies suggested that transmission events could originate from schools. This study aimed to evaluate early-warning methods for identifying asymptomatic COVID-19 cases by implementing screening programs in schools. This study was conducted between September 2021 and May 2023, employing a rotation-screening plan for COVID-19 detection on a sample of students aged 14 to 19 years attending secondary schools in the regions of Tuscany, Veneto, Apulia and Friuli-Venezia Giulia. The schools were divided into two groups: experimental and control, with a ratio of 1:2. Two types of molecular salivary tests for SARS-CoV-2 were used to conduct the screening. This study included 16 experimental schools and 32 control schools. Out of 2527 subjects, 11,475 swabs were administrated, with 9177 tests deemed valid for analysis (a 20% loss of tests). Among these, 89 subjects (3.5%) tested positive. In control schools, 1895 subjects (6.5%) tested positive for SARS-CoV-2. This study recorded peaks in infections during the winter and autumn months, consistent with patterns observed in the general population. Beginning in September 2022, a shift occurred, with 2.6% of positive cases reported in the case schools compared to 0.3% in the control schools. Initially, most cases of COVID-19 were detected in the control schools; however, as the pandemic emergency phase concluded, cases were primarily identified through active screening in experimental schools. Although student participation in the active screening campaign was low during the project’s extension phase, this approach was efficacious in the early identification of positive cases.

Background:Accurate detection of β-lactam resistance genes in bloodstream infections is critical for guiding antimicrobial therapy. This study evaluates the Alifax Gram-negative resistance (GNR) microchip assay for detecting β-lactam resistance genes directly from positive blood cultures (PBCs) for Gram-negative (GN) bacteria, including Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii. Methods:Simulated (n=146) and clinical (n=106) GN-PBC samples were tested for bla KPC, bla VIM, bla NDM, bla IMP, bla OXA-23-like, bla OXA-48-like, bla SHV-ESBL, bla CTX-M-1/9 group, and bla CMY-2-like genes using the GNR microchip assay. Whole-genome sequencing (WGS) served as the reference assay for simulated samples and, selectively, for clinical samples. The bioMérieux BioFire Blood Culture Identification 2 (BCID2) panel assay was used as a comparator for clinical samples. Results:The GNR microchip assay correctly identified 203 (99.5%) of 204 β-lactam resistance genes in simulated samples. One sample tested false negative for a bla SHV-ESBL gene but true positive for a bla KPC gene. In clinical samples, GNR results were concordant with BCID2 for 113 (100%) of 113 genes included in both assays. Additionally, the GNR assay detected bla CMY-2 -like (n=6), bla OXA-23-like (n=5), and bla SHV-ESBL (n=2), which are not targeted by BCID2, all confirmed by WGS. In two β-lactam-resistant P. aeruginosa samples but negative by the GNR assay, WGS confirmed the absence of acquired β-lactam resistance genes, suggesting alternative resistance mechanisms. Conclusion:The GNR microchip assay demonstrated high concordance and broader β-lactam resistance gene coverage compared to BCID2, supporting its potential role in routine diagnostics. Further validation in larger, prospective studies is warranted.

BACKGROUND:Blood culture (BC) remains the cornerstone for diagnosis of bloodstream infections (BSI), but the long turn-around time (TAT) hampers timely selection of appropriate chemotherapy. Novel molecular approaches have been developed to provide faster results but are also affected by limitations. We developed a analytical workflow named LC-WGS (Whole-Genome Sequencing of Liquid Colony) for rapid whole-genome sequencing-based diagnosis of BSI, evaluating its accuracy performance over standard of care (SoC) diagnostic procedures. METHODS:A total of 85 prospectively collected positive BC were processed in parallel with SoC (subculturing, identification by MALDI-ToF, antimicrobial susceptibility testing by reference broth microdilution, usage of syndromic panels) and LC-WGS, which relied on automated purification of microbial cells (Qvella FAST system, Qvella Corp.), DNA purification, and real-time sequencing with the Oxford Nanopore MinION. A streamlined analysis pipeline was designed for pathogen identification (Kraken2), detection of resistance markers (KmerResistance, AMRFinderPlus), virulome profiling (abricate, VFDB), phylogenetic analysis (snippy, IQ-TREE), and pathogen subtyping (Meningotype). FINDINGS:Compared with SoC, LC-WGS returned accurate species-level identification for 98% (65/66) of monomicrobial and 88% (14/16) of polymicrobial BCs, with a TAT as short as ∼2·6 h. Accurate resistome profiling (allelic variants) was achieved for 94% (58/62) of the most clinically-relevant resistance profiles in ∼4·2 h. In silico serotying (Neisseria meningitidis), virulotyping (Escherichia coli, Klebsiella pneumoniae) and comparative phylogenomics for outbreak investigation (K. pneumoniae) proved also feasible. INTERPRETATION:In this proof-of-concept study, we proved that diagnosis of BSI can be significantly shortened using an optimised workflow based on real-time sequencing, providing rapid, actionable clinical microbiological data in support of timely selection of appropriate chemotherapy. LC-WGS proved also useful as molecular epidemiology tool for public health and infection control applications. FUNDING:This study was partially supported by an investigator-initiated grant from Qvella Corporation.

Background/Objectives: The emergence and spread of carbapenemase-producing Enterobacterales (CPE) represents a significant challenge prompting the need to optimize diagnostic tools to detect CPE carriers. The Xpert® Carba-R assay (Cepheid, Sunnyvale, CA, USA), a Real-Time PCR-based test, can detect the blaKPC, blaVIM, blaOXA-48, blaNDM and blaIMP carbapenemase genes directly from rectal swabs. This study assessed the performance of the Xpert® Carba-R assay using FecalSwab™ (Copan, Brescia, Italy), a liquid-based collection device. Methods: The first part of the study aimed to establish the FecalSwabTM volume which gave the most similar Ct values to those obtained by the Transystem™ double swab (Copan). The best volume was then used to assess the limit of detection (LoD) for each target and compare the accuracy of different FecalSwabTM storage conditions (room temperature or 4 °C after 16 h compared to T0). Results: The results indicated that using 200 µL of the FecalSwab™ medium provided reliable Ct values, with the lowest number of invalid samples compared to traditional methods. The average LoDs for different carbapenemases ranged from 4.7 × 103 to 6.8 × 103 CFU/mL. FecalSwab™ showed a better performance after 16 h at room temperature compared to storage at 4 °C. Conclusions: This study supports single sampling with the FecalSwab™ medium for both molecular and cultural methods, for the potential optimization of CPE screening.

Antimicrobial resistance (AMR) has emerged as one of the major challenges for human health, with a remarkable burden of mortality, morbidity, and healthcare-associated costs [...]

BACKGROUND:The case confirmation of CL relies on the direct demonstration of the parasite in clinical specimens from skin tissues. Despite most research efforts focusing on biopsy samples as the preferred diagnostic specimen for the detection of Leishmania spp., the use of non-invasive sampling, such as cutaneous swabs, combined with the use of molecular assays, has shown promising results. METHODS:We conducted a retrospective study aimed at comparing the performance of different invasive and non-invasive diagnostic techniques, employed for the diagnosis of CL, in an Italian tertiary care center. RESULTS:We observed 29 cases of CL between 2008 and June 2024. Considering the demonstration of Leishmania spp. on culture, biopsy PCR, histology, or smear microscopy as the reference diagnostic test for CL, molecular assays on cutaneous swabs showed a sensitivity of 100% (95% C.I. 73.5-100). Overall, PCR performed on swab specimens allowed for the detection of three cases that biopsy histology (in two cases) and microscopic examination of cutaneous smear (in three cases) would have failed to identify. CONCLUSION:Non-invasive swab sampling, combined with molecular analysis, can be a valuable tool for a more accessible and patient-friendly diagnostic approach for CL. Should our preliminary results be confirmed, this test could become the first-line diagnostic tool for CL, reserving biopsy as a second-level test or for cases in which the differential diagnosis includes malignancy or other concerning diseases. Further studies aimed at defining the efficiency of this diagnostic method and providing standardized diagnostic protocols would be needed to provide stronger evidence supporting its recommendation.

Blood culture (BC) remains the reference diagnostic tool for bloodstream infections but is hampered by long turn-around time (TAT). This study evaluated the Vitek® Reveal™ (VR) system for rapid antimicrobial susceptibility testing (AST) with 72 cases of monomicrobial BCs (55 Enterobacterales, 12 Pseudomonas aeruginosa and 5 Acinetobacter baumannii), including isolates producing carbapenemases and/or extended-spectrum β-lactamases. VR returned AST results with a mean TAT of 5.4 h. Compared to a conventional workflow based on broth microdilution, VR exhibited essential agreement (EA) and category agreement (CA) >90 % in most cases, except with meropenem for Enterobacterales (CA, 85.5 %), piperacillin/tazobactam for P. aeruginosa (EA, 83.3 %), and trimethoprim/sulfamethoxazole for A. baumannii (CA and EA, 80 %). Bias exhibited an underestimation trend with ceftazidime/avibactam (-78.9 %) and ceftazidime (-50 %) for Enterobacterales and P. aeruginosa, respectively. Overall, VR appears an interesting tool to decrease TAT of the BC workflow, although further evaluation with some antibiotic-pathogen combinations would be warranted.

Wastewater-based epidemiology has proved to be a suitable approach for tracking the spread of epidemic agents including SARS-CoV-2 RNA. Different protocols have been developed for quantitative detection of SARS-CoV-2 RNA from wastewater samples, but little is known on their performance. In this study we compared three protocols based on Reverse Transcription Real Time-PCR (RT-PCR) and one based on Droplet Digital PCR (ddPCR) for SARS-CoV-2 RNA detection from 35 wastewater samples. Overall, SARS-CoV-2 RNA was detected by at least one method in 85.7% of samples, while 51.4%, 22.8% and 8.6% resulted positive with two, three or all four methods, respectively. Protocols based on commercial RT-PCR assays and on Droplet Digital PCR showed an overall higher sensitivity vs. an in-house assay. The use of more than one system, targeting different genes, could be helpful to increase detection sensitivity.

Antimicrobial susceptibility testing (AST) from blood culture (BC) may take several days, limiting the eventual impact on antimicrobial stewardship. Hence, rapid AST systems represent a valuable support in shorting the time-to-response. In this work, the Quantamatrix dRASTTM system (dRAST) was evaluated for rapid AST on 100 monomicrobial BCs (50 Gram-negatives and 50 Gram-positives), including several isolates with clinically relevant resistance mechanisms. AST results were provided in 6-hours, on average. Compared to Micronaut (Merlin) system based on broth microdilution, dRAST exhibited an overall categorical agreement of 92.5 %, essential agreement of 89.0 %, and mean bias of 15.9 %. Category overestimation (potentially leading to unnecessary high-dosage treatment or to exclude active agents) and category underestimation (potentially leading to underdosing or using ineffective agents) were observed in 4.3 % and 3.1 % of cases, respectively. Even though several issues were reported, results confirmed the potential contribution of dRAST to shorten the BCs clinical microbiology workflow and management.

Detection of SARS-CoV-2 in bronchoalveolar lavage (BAL) is considered as a promising alternative method to detect COVID-19 infection. STANDARD™ M10 SARS-CoV-2 assay on 150 negative and 50 positives BAL samples for SARS-CoV-2 showed 96 % sensitivity, 100 % specificity compared to Allplex™ SARS-CoV-2 assay and a 31.25 genomic copies/mL limit of detection.

The emergence and global dissemination of carbapenemaseproducing Gram-negative bacteria (CPGNB) pose a significant public health threat due to the limited therapeutic options for CPGNB infections, which are associated with high mortality rates and a considerable economic and societal burden. 1 Early identification of CPGNB is useful for timely infection prevention and control measures and therapeutic stewardship, and identification of the resistance mechanisms has become essential following the availability of several new antimicrobial agents with variable coverage depending on resistance mechanism. 2 Lateral flow immunoassays (LFIAs) are rapid (15-30 min) systems for detection of clinically relevant resistance mechanisms, including carbapenemases.LFIAs require limited technical expertise in the absence of expensive equipment, and are generally less expensive than genotypic methods. 3,4 Moreover, unlike the latter, LFIA systems detect proteins and are not affected by false positivity in case of silenced genes. 4 However, false negativity may also occur with LFIA systems in case of low level of production of the target protein (e.g. with weakly expressed MBLs), 5,6 or of amino acid changes in the epitopes of the target protein recognized by the antibody probe used by the system (e.g.changes at position 179 in the Ω-loop of KPC carbapenemase variants, which confer resistance to avibactam and exhibit impaired carbapenemase activity). [7][[9][10][11] In this work we evaluated three commercial LFIA systems for detection of the five most common families of carbapenemases encountered among CPGNB, namely the KPC/IMP/NDM/VIM/ OXA-48 Combo Test Kit (KINVO, Medomics Medical Technology, Jiangsu, China), the O.K.