Search Results (46)

Companion animals are increasingly recognized as carriers of antimicrobial-resistant Escherichia coli, yet genomic data from clinically healthy dogs in Latin America remain scarce. We characterized 13 genetically non-redundant multidrug-resistant E. coli isolates selected from 224 resistant E. coli isolates recovered in a previous surveillance study of 600 clinically healthy household dogs in the Metropolitan Region of Chile (2021–2022). Antimicrobial susceptibility testing was performed using VITEK2 and interpreted according to Clinical and Laboratory Standards Institute criteria, and whole-genome sequencing was used to identify resistance genes, virulence-associated traits, plasmid replicons, phylogroups, sequence types, and ST131 subclades. All isolates were multidrug resistant and showed high resistance to ampicillin and cephalexin (13/13, 100%), ciprofloxacin (13/13, 100%), cefpodoxime (11/13, 84.6%), cefovecin (10/13, 76.9%), and ceftiofur (10/13, 76.9%). An extended-spectrum β-lactamase phenotype was observed in 9/13 isolates (69.2%). bla_CTX-M genes were detected in 7/13 isolates (53.8%) and bla_CMY-2 in 3/13 isolates (23.1%), whereas IncF-associated plasmid replicons predominated. The isolates belonged to diverse lineages, including ST131, ST744, ST1196, and ST1011. The two ST131 isolates belonged to phylogroup B2, carried bla_CTX-M variants, and were assigned to the C2/H30Rx and C1-M27 subclades. These findings support further consideration of companion animals in genomic antimicrobial resistance surveillance under a One Health framework. Full article

Background/Objectives: Given the ongoing threat of antimicrobial resistance, the identification and characterization of multidrug-resistant isolates are essential. An increase in antimicrobial-resistant bacteria has been reported in Romania, but national data are still scarce. This study aimed to evaluate beta-lactamase-producing Gram-negative bacteria (GNB) isolated over two years at a Romanian nephrology hospital, while comparing carbapenemase detection phenotypic methods. Methods: Gram-negative bacterial isolates collected between January 2022 and May 2024 that met antimicrobial resistance screening criteria were evaluated. After identification, extensive disk diffusion antibiograms were performed, read, and interpreted, complemented by testing on cloxacillin/oxacillin-supplemented Mueller–Hinton agar. The colistin minimum inhibitory concentration (MIC) was not assessed, and aztreonam–avibactam was not tested for Enterobacterales. For non-fermenter GNB, the colistin MIC was determined. Phenotypic carbapenemase production tests were performed for all strains (BlueCarba Test, CIM, mCIM, zCIM, and rCIM). Carbapenemase detection immunochromatographic tests were performed for a set of strains. Results: Among the 397 evaluated strains, 335 (84.38%) were Enterobacterales and 62 (15.62%) non-fermenter GNB, showing high antimicrobial resistance levels. Of these, 188 (47.35%) were Klebsiella pneumoniae; 139/188 (73.93%) showed carbapenem resistance and carbapenemase production; 49/188 (26.06%) produced two carbapenemases; and 45/188 (23.93%) presented resistance to all tested antimicrobials. MALDI-TOF identified 28 KPC-producing K. pneumoniae strains. Lateral flow assays revealed NDM, VIM, KPC, and OXA-48-like enzymes in 48 of 56 tested Enterobacterales; 12/48 strains produced two carbapenemases. Of the 62 non-fermenter GNB, 33 were Pseudomonas spp. and 20 Acinetobacter baumannii; one Pseudomonas spp. was susceptible only to colistin and seven only to cefiderocol; four A. baumannii were susceptible only to colistin and three only to cefiderocol. Lateral flow assays detected VIM or IMP enzymes in 13/33 Pseudomonas spp. and OXA-23 and/or OXA-40/-58 enzymes in all 20 A. baumannii. Conclusions: Among the evaluated strains, many showed resistance to multiple antimicrobial classes. Furthermore, strains co-producing two carbapenemases were identified. Full article

Background: Multi-criteria decision analysis (MCDA) has become a dominant approach for glacier hazard susceptibility mapping, widely used to support risk management and climate adaptation planning. However, despite its widespread adoption, the role of MCDA outputs remains conceptually ambiguous: hazard classifications are often interpreted as predictive representations of risk, even though they are derived from preference-dependent decision models. This raises a critical but underexamined question regarding the reliability of MCDA-based glacier hazard assessments. This issue becomes particularly relevant in the current transition toward data-driven and artificial intelligence (AI)-based approaches for hazard modelling, where similar challenges of interpretability, validation, and reliability arise. Methods: To address this issue, we conducted a systematic literature review following the PRISMA 2020 protocol, analysing peer-reviewed studies published between 2015 and 2025. After screening 571 records, 60 studies were included. Data were extracted using a structured framework and synthesised through quantitative descriptive analysis and qualitative assessment of modelling practices, including method selection, criteria weighting, uncertainty treatment, validation, and geographical distribution. This study conducts a structured methodological audit—not a catalogue—of multi-criteria decision analysis (MCDA) applications in glacier hazard susceptibility mapping. Results: The analysis reveals a consistent methodological pattern. The Analytic Hierarchy Process (AHP) dominates current practice (36/60 studies, 60%), typically implemented through GIS-based weighted overlay with expert-derived weights. Critically, 80% of studies (48/60) derive criteria weights exclusively from expert judgement, with no data-driven calibration or sensitivity testing of subjective inputs. This epistemic reliance on unstructured or semi-structured expert elicitation, presented without robustness analysis, forms a central concern of this review. Moreover, empirical validation is limited: only 21/60 studies (35.0%) report quantitative performance metrics. Uncertainty and robustness analyses are rarely conducted, and most studies rely on single-model configurations without comparative evaluation. Despite these limitations, the resulting hazard maps are frequently presented as objective spatial predictions. The evidence base is also geographically concentrated, with 48/60 studies (80.0%) located in High Mountain Asia. Conclusions: The findings indicate a systematic mismatch between how MCDA-based hazard maps are constructed and how they are interpreted. In most cases, MCDA functions as a decision-structuring framework rather than a validated predictive model, yet its outputs are commonly treated as predictive evidence. This gap has important implications for the use of such models in risk management and climate adaptation, particularly in the emerging context of AI-driven hazard modelling, where issues of model validation, interpretability, and reliability become even more critical. Advancing the field requires explicit validation against observed events, systematic robustness and sensitivity analysis, transparent uncertainty modelling, and comparative evaluation of alternative or hybrid decision frameworks. Full article

Background: The role of bacterial infection in canine extrahepatic biliary disease remains inconsistently characterized. This retrospective case series evaluated associations between ultrasonographic gallbladder findings and bile culture results in dogs. Materials and Methods: Sixty-seven dogs were screened between March 2024 and December 2025, of which 49 met inclusion criteria (clinical, biochemical, and ultrasonographic evidence of gallbladder disease) and underwent bile sampling by ultrasound-guided cholecystocentesis (n = 45) or intraoperative collection (n = 4). Samples were cultured aerobically and anaerobically; isolates identified by MALDI-TOF MS were further tested for in vitro antimicrobial susceptibility using Kirby–Bauer disk diffusion method. Results: Among the 49 included dogs, ultrasonography identified cholecystitis (75.5%), cholelithiasis (16.3%), and biliary mucocele (8.2%). Bile cultures were positive in 43/49 dogs (87.8%), yielding only aerobic bacteria. Escherichia coli (46.5%) and coagulase-positive Staphylococci (30.2%) were most frequently isolated organisms. However, these findings should be interpreted cautiously due to small subgroup sizes and the exploratory nature of the analysis. Conclusions: In this selected referral population, bacterial isolates were frequently recovered from bile samples, particularly in dogs with cholecystitis. Prospective studies involving larger populations are warranted to confirm these results, define the bacterial prevalence and clinical significance of bacterial colonization or infection, and further refine evidence-based diagnostic and treatment strategies for dogs with extrahepatic biliary disease. Full article

Background: Acinetobacter baumannii is a non-fermenting Gram-negative bacillus responsible for severe nosocomial infections, particularly in intensive care units (ICUs). The increasing prevalence of multidrug-resistant (MDR) and carbapenem-resistant A. baumannii (CRAB) strains has become a significant challenge for infection control and antimicrobial therapy worldwide. Objectives: This study aimed to analyze the antimicrobial susceptibility patterns of clinical A. baumannii isolates recovered from a multi-profile hospital in years 2020–2024 in Lodz, Poland. Methods: Clinical isolates from various specimen types (blood, urine, wound swabs, biopsies, sputum, and bronchoalveolar lavage fluid) were obtained during routine microbiological diagnostics. Identification was performed using MALDI-TOF MS. Antimicrobial susceptibility testing (AST) was conducted using the automated VITEK^®2 system with EUCAST/CLSI interpretive criteria. Minimum inhibitory concentrations (MICs) for colistin were determined by broth microdilution. Carbapenemase production was assessed using the Carbapenem Inactivation Method (CIM) and immunochromatographic assays for OXA-23, OXA-40/58, and NDM detection. Results: A total of 244 A. baumannii isolates were recovered over the study period. Susceptibility to carbapenems (meropenem, imipenem) declined markedly, with resistance exceeding 90% by 2023–2024. Aminoglycosides exhibited variable activity, with gentamicin demonstrating the highest susceptibility rates (up to 88% in 2022). Resistance to ceftazidime and cefepime remained consistently high (>90% in 2023–2024). No fully susceptible isolates were identified for ciprofloxacin. Conclusions: The high prevalence of CRAB strains highlights the urgent need for effective infection control measures, optimized antimicrobial stewardship, and consideration of novel treatment options in the clinical setting. Full article

Background/Objectives: Accurate determination of colistin (COL) in vitro activity against carbapenem-resistant Acinetobacter baumannii complex (CRAB) isolates remains challenging, as the reference broth microdilution (BMD) method is labor-intensive and not routinely implemented in most clinical laboratories. Semi-automated susceptibility methods for colistin in the clinical laboratory require validation. The present study evaluated the performance characteristics of the recently introduced Vitek^®2 card AST-N440 for COL antimicrobial susceptibility testing (AST) on CRAB isolates compared with a BMD-based reference method (ComASP Colistin). Methods: A total of 176 single-patient CRAB isolates from two distinct tertiary Greek hospitals between 2024 and 2025 were included. COL susceptibility testing was performed using Vitek^®2 AST-N440 and compared with BMD. Minimum inhibitory concentrations (MICs) were interpreted according to EUCAST breakpoints. Method performance was evaluated by calculating categorical (CA) and essential agreement (EA), sensitivity, specificity, positive and negative predictive values (PPV/NPV), and major (ME) and very major error rates (VME) according to ISO 20776-2. Results: Compared with BMD, AST-N440 showed a sensitivity of 89.6% and a specificity of 62.3%, with a PPV and NPV of 81.7% and 76.0%, respectively. The CA (80.1%) and the EA (46.0%) were below ISO acceptance criteria. The VME rate was 10.4%, and the ME rate 37.7%. Identical MIC values were observed in 25.0% of the isolates, while Vitek^®2 reported lower and higher MIC values than BMD in 46.6% and 28.4% of isolates, respectively. Conclusions: The Vitek^®2 AST-N440 card performed suboptimally for COL susceptibility testing in CRAB isolates. Further validation of automated systems for COL AST is needed. Full article

Background/Objectives: Urinary tract infections (UTIs) are among the most common bacterial infections and represent a major source of antimicrobial use. Increasing antimicrobial resistance among uropathogens, particularly the emergence of extended-spectrum beta-lactamase (ESBL)-producing organisms, complicates empiric treatment strategies. ESBL-producing organisms are clinically relevant because they are frequently associated with multidrug resistance and significantly limit empiric antimicrobial treatment options in urinary tract infections. The study period starting in 2019 was selected to reflect contemporary resistance patterns and to ensure consistency with the updated EUCAST antimicrobial susceptibility interpretation criteria introduced at that time. This study aimed to characterize antimicrobial resistance patterns among uropathogens isolated from lower UTIs and to identify independent predictors of antimicrobial resistance using isolate-level analyses. Methods: This retrospective observational study included 1470 patients and isolates with clinically suspected lower UTIs who underwent urine culture and antimicrobial susceptibility testing between 2019 and 2024 at a single clinical center. Antimicrobial susceptibility was interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria, and ESBL production was assessed among Gram-negative (GN) isolates. Multivariable generalized estimating equation (GEE) logistic regression models accounting for patient clustering were used to identify predictors of resistance. Results: A total of 1470 patients and isolates were included. Escherichia coli was the most frequent uropathogen (66.0%), followed by Klebsiella pneumoniae and Enterococcus faecalis. Among Gram-negative isolates, 17.3% were ESBL-positive. Resistance rates were highest for ciprofloxacin (35.4%) and trimethoprim/sulfamethoxazole (31.7%), while fosfomycin and nitrofurantoin retained high activity against E. coli. In multivariable analyses, ESBL production was the strongest independent predictor of resistance to several antimicrobials, including ciprofloxacin (aOR 9.83), amoxicillin/clavulanic acid (aOR 3.22), trimethoprim/sulfamethoxazole (aOR 2.89), and cefotaxime (aOR 1337). Pathogen identity was also independently associated with resistance. Conclusions: Antimicrobial resistance among uropathogens was heterogeneous and predominantly driven by pathogen identity and ESBL production. ESBL status emerged as the most consistent and powerful predictor of resistance across multiple antimicrobials, underscoring its clinical relevance for empiric treatment decisions and antimicrobial stewardship in urinary tract infections. Full article

Background: Klebsiella pneumoniae is a World Health Organization-listed critical priority pathogen and a major cause of healthcare-associated infections, driven by the global emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) lineages and their alarming convergence with hypervirulence. Methods: In this study, 152 clinical specimens, including urine, blood, pus, wound swabs, and respiratory samples, were collected from tertiary care hospitals in Peshawar, Pakistan. Standard microbiological and biochemical methods identified 55 K. pneumoniae isolates. Antimicrobial susceptibility testing (AST) was performed using the Kirby–Bauer disk diffusion and broth microdilution methods, with results interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines. MDR and XDR phenotypes were defined based on European Centre for Disease Prevention and Control (ECDC) criteria. Whole-genome sequencing (WGS) was conducted on 16 phenotypically confirmed MDR/XDR isolates, followed by comprehensive bioinformatic analyses to characterize sequence types (STs), acquired antimicrobial resistance genes, resistance-associated chromosomal mutations, virulence determinants, plasmid replicons, and phylogenetic relationships. Results: Among 55 confirmed K. pneumoniae isolates, 19 (34.5%) were classified as MDR and 10 (18.2%) as XDR. WGS revealed substantial genomic heterogeneity, identifying 11 distinct STs, with ST39 being the most prevalent. Resistance to multiple antibiotic classes was mediated by the combined presence of plasmid-borne carbapenemases and extended-spectrum β-lactamases, alongside chromosomal mutations affecting outer membrane porins (OmpK35/OmpK36), fluoroquinolone targets (gyrA/parC), efflux regulation (ramR, marR), and lipid A modification pathways associated with colistin resistance (mgrB, pmrA/pmrB, arnC, crrB). IncF-family plasmids predominated and frequently co-occurred with additional resistance-associated replicons. Notably, one isolate exhibited an expanded virulence gene repertoire, including multiple siderophore systems and a complete type II secretion system, consistent with a hypervirulence-associated genomic profile. Phylogenetic analyses demonstrated close relatedness to international lineages from Asia, the Middle East, and Europe, indicating regional and transnational dissemination. Conclusions: This study highlights the complex interplay between plasmid-mediated gene acquisition and chromosomal adaptive mutations driving MDR and XDR phenotypes in K. pneumoniae circulating in Peshawar, Pakistan. The identification of hypervirulence-associated genetic features within an MDR background underscores the growing threat posed by convergent lineages and emphasizes the need for sustained WGS-based surveillance to inform infection control and antimicrobial stewardship strategies. Full article

Background/Objetives: The objective of this study was to determine the in vitro susceptibility profiles of clinical Clostridioides difficile isolates to metronidazole (MTZ), vancomycin (VAN), fidaxomicin (FDX), tigecycline (TGC), and eravacycline (ERV) in a multicenter Spanish cohort, and to evaluate their association with clinical factors. Methods: Strains were obtained from prospectively included patients in the ICD-ANCRAID-SEICV cohort (ClinicalTrials.gov ID: NCT04801862) in Andalucía and the Valencian Community between 1 January 2020 and 30 April 2023. Antimicrobial susceptibility testing was performed using E-test for MTZ, VAN, TGC, and ERV, and agar dilution for FDX. Results: The results were interpreted following EUCAST clinical breakpoints and ECOFF criteria. A total of 107 patients were included (median age 70 years; 65.4% women). Nearly half of the cases were community-acquired, 30% nosocomial, and the remainder healthcare-associated. Most infections were non-severe, and 32.7% experienced recurrence. Overall resistance levels were low: VAN and TGC each showed resistance in 2.8% of isolates, followed by MTZ (1.9%). Only one isolate was resistant to FDX (0.9%), and none to ERV. MIC90 values were low for all agents. Some resistant isolates displayed co-resistance and were recovered from patients with prior antibiotic exposure. Among the seven patients carrying resistant strains, most were women, and the cases were predominantly community-acquired. Clinical characteristics, including age, comorbidity, infection origin, and severity, did not differ from those with susceptible isolates. All patients achieved clinical cure without recurrent infection. No association was found between elevated MIC values and recurrence or greater severity. Conclusions: FDX and ERV demonstrated excellent in vitro activity. Resistance to MTZ, VAN, and TGC was uncommon but detectable. Findings highlight the need for continued antimicrobial resistance surveillance and evaluation of its potential clinical impact. Full article

Background: Vancomycin is a critically important antimicrobial in human medicine, and vancomycin-non-susceptible enterococci represent a One Health concern when animal reservoirs contribute to the wider resistance ecology. We aimed to characterize vancomycin non-susceptibility among poultry-derived Enterococcus spp. from Hungary, using a combined phenotypic–genomic approach. Methods: Following a phenotypic pre-screen with antimicrobials authorized for poultry, 218 isolates with elevated minimum inhibitory concentrations (MICs) were selected for extended broth microdilution testing including vancomycin. Vancomycin susceptibility was interpreted using Clinical and Laboratory Standards Institute (CLSI) clinical breakpoints and European Committee on Antimicrobial Susceptibility Testing (EUCAST) epidemiological cut-off values (ECOFFs). Whole-genome sequencing was performed on a targeted multidrug resistant (MDR) subset (n = 42), enriched for elevated or borderline vancomycin MICs and stratified by region and host species (chicken, turkey), and resistance determinants were annotated against the Comprehensive Antibiotic Resistance Database (CARD) using stringent similarity/coverage thresholds. Results: Among the 218 pre-screened isolates (126 from chickens; 92 from turkeys), 196 (89.9%) met MDR criteria. For vancomycin, 15.6% of isolates were resistant and 9.2% intermediate by CLSI, while EUCAST ECOFF classification placed 34.9% in the non-wild-type group. The vancomycin MIC distribution was right shifted, with high-end MICs observed. In the sequenced subset, vancomycin-associated determinants consistent with the vanC pathway (including regulatory and auxiliary components) were detected in five isolates. Beyond vancomycin-related determinants, the WGS subset harbored common resistance genes consistent with the observed multidrug-resistant phenotypes. Conclusions: Vancomycin non-susceptibility was detected among pre-screened poultry-derived Enterococcus isolates in Hungary, and genomic analysis revealed vanC-associated and other peptide antibiotic resistance signatures. These findings support targeted One Health surveillance integrating MIC distributions with genomic resistance determinants in food animal reservoirs. Full article

Burkholderia pseudomallei, the causative agent of melioidosis, is endemic in Sarawak, Malaysian Borneo, where it is represented by a unique gentamicin-susceptible population. Despite trimethoprim-sulfamethoxazole (co-trimoxazole) being the cornerstone of eradication therapy, emerging reports of elevated minimum inhibitory concentrations (MICs) among Sarawak isolates have raised concerns over its clinical efficacy. We performed a retrospective and comprehensive antibiotic susceptibility assessment of clinical B. pseudomallei isolates from hospitals across Sarawak. Susceptibility to trimethoprim-sulfamethoxazole was determined using disk diffusion and the E-test, interpreted by both CLSI and EUCAST guidelines. Resistance to the individual components, trimethoprim and sulfamethoxazole, was characterized by broth microdilution. The results demonstrated a high prevalence of trimethoprim-sulfamethoxazole susceptibility, with 96.3% of isolates susceptible by CLSI criteria and 97.6% by EUCAST criteria. Interestingly, broth microdilution revealed that resistance to trimethoprim and sulfamethoxazole individually did not confer resistance to the synergistic combination. Our analysis validated CLSI guidelines as the most reliable standard for antimicrobial resistance surveillance in this region. This study provides evidence that trimethoprim-sulfamethoxazole remains effective for melioidosis treatment in Sarawak, offering crucial reassurance to clinicians. The paradoxical finding of susceptibility to the drug combination despite resistance to its individual components underscores the critical importance of the synergistic activity of trimethoprim-sulfamethoxazole and highlights the need for further investigation into the molecular basis of resistance in this distinct B. pseudomallei population. Full article

Background: Stenotrophomonas maltophilia is an intrinsically multidrug-resistant, biofilm- forming, non-fermenter increasingly implicated in hospital-acquired infections. Evidence from non-cystic fibrosis populations, especially in the Middle East, remains sparse. Methods: We conducted a retrospective observational cohort study at a tertiary academic center (Al-Khobar, Saudi Arabia) spanning 1 May 2001–30 April 2023. Hospitalized adults (≥18 years) with culture-confirmed, clinically diagnosed S. maltophilia infection and ≥72 h of antibiotic therapy were included. The primary outcome was all-cause mortality (14-day, 30-day, 1-year). Secondary outcomes were clinical response, microbiological eradication, and infection recurrence. Predictors of 30-day mortality were assessed using multivariable logistic regression; 14-day mortality was analyzed by Kaplan–Meier/log-rank according to susceptibility-guided versus alternative therapy. Results: Of 539 patients with positive cultures, 436 met the inclusion criteria. Mean age was 60.5 ± 19.3 years; 62.2% were male. Most infections were hospital-acquired (92.9%); pneumonia composed 64.7% and bloodstream infection 15.4%. Polymicrobial growth occurred in 55.5% (predominantly Gram-negative co-isolation). Susceptibility was 95.1% to trimethoprim–sulfamethoxazole, 76.4% to levofloxacin, and 43.6% to ceftazidime. Mortality at 14 days, 30 days, and 1 year was 22.8%, 37.9%, and 57.2%, respectively. On multivariable modelling, intensive care unit (ICU) admission, leukocytosis, neutrophilia, anemia, and thrombocytopenia independently predicted 30-day mortality. Susceptibility-guided therapy was associated with improved 14-day survival (log-rank p = 0.033). Conclusions: In this large, long-running non-cystic fibrosis cohort, host acuity and early alignment of treatment to susceptibility data were dominant drivers of outcome. High polymicrobial burden and limited reliably active agents underscore the need for meticulous stewardship, robust infection prevention, and cautious interpretation of S. maltophilia antimicrobial susceptibility testing. Full article

Background/Objectives: Fingernail onychomycosis differs etiologically and epidemiologically from toenail infections and is frequently complicated by colonization and mixed growth. Reliable interpretation of microscopy–culture correlations is essential for avoiding overdiagnosis and guiding therapy. This study aimed to characterize the diagnostic structure, species distribution, and antifungal susceptibility patterns of fingernail onychomycosis in a large routine-laboratory cohort, and to evaluate the performance of a five-tier operational classification integrating microscopy and semi-quantitative culture. Methods: Laboratory records from 1075 patients with clinically suspected fingernail onychomycosis (including nail and periungual samples) were analyzed retrospectively (2017–2024). Direct microscopy with calcofluor white, semi-quantitative culture, and MALDI-TOF MS identification were performed. Cases were categorized based on predefined criteria combining microscopic elements with colony quantity and purity. Species distribution, age–sex patterns, diagnostic concordance between microscopy and culture, and results of EUCAST broth microdilution testing for selected yeasts were assessed. Results: The overall proportion of mycologically positive cases was similar in women and men, although age-dependent patterns differed. Microscopic findings correlated with culture outcomes, with hyphae predicting dermatophytes, yeast cells predicting ascomycetous yeasts, and negative slides aligning with the absence of growth. Yeasts predominated (Candida parapsilosis 30.9%, C. albicans 18.5%), dermatophytes were mainly Trichophyton rubrum, and molds were uncommon. Periungual swabs showed species distributions closely matching those from nail samples and demonstrated high analytical concordance. EUCAST MICs revealed species-dependent variation, including elevated amorolfine MICs in C. parapsilosis and reduced fluconazole activity in Wickerhamomyces pararugosa. Conclusions: Fingernail onychomycosis in this cohort was predominantly yeast-associated, with predictable microscopy–culture relationships and distinct age–sex patterns. The five-tier operational framework improved classification of infection versus colonization, and is proposed as a preliminary tool requiring clinical validation, while contemporary MIC data highlighted clinically relevant interspecies differences. The absence of clinical correlation data (symptoms, severity, treatment history) remains the primary limitation, preventing definitive distinction between infection and colonization in all cases. Full article

Background/Objectives: Malignant hyperthermia (MH) is a life-threatening pharmacogenetic disorder of skeletal muscle calcium regulation and commonly associated with pathogenic variants in the RYR1 gene. Interpretation of rare RYR1 variants remains challenging, particularly when classified as variants of uncertain significance (VUS). This study describes the clinical, functional, and genetic evaluation of a patient with suspected MH susceptibility carrying a rare RYR1 mutation. Methods: We report a retrospective case evaluation of a 32-year-old female referred for MH assessment following a prior peri-operative hypermetabolic event. Clinical records were reviewed, and MH susceptibility was assessed using the caffeine–halothane contracture test (CHCT). Genetic testing was performed using a targeted MH susceptibility gene panel, including RYR1, CACNA1S, and STAC3. Variant classification was conducted following American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines. Results: The patient demonstrated a positive CHCT, consistent with MH susceptibility. Genetic analysis identified a rare heterozygous RYR1 missense variant. No pathogenic or likely pathogenic variants were detected in CACNA1S or STAC3. Based on ACMG/AMP criteria, the RYR1 p.Gln474His variant is currently classified as a VUS. However, its localization within the N-terminal regulatory region of RyR1 and concordance with abnormal CHCT findings provide supportive functional context. Conclusions: This case underscores the importance of integrating clinical history, functional contracture testing, and genetic data in the evaluation of MH susceptibility. While functional findings may support biological plausibility, definitive pathogenic classification of rare RYR1 variants requires additional segregation data or mechanistic studies. Full article

Background: Rapid antimicrobial susceptibility testing (RAST) allows early detection of resistance directly from positive blood cultures, potentially improving outcomes in bloodstream infections (BSIs). We evaluated the performance of EUCAST RAST for Gram-negative ESKAPEEc pathogens and characterized carbapenemase genes in carbapenem-resistant Klebsiella pneumoniae (CRKP). Methods: A total of 354 positive blood cultures were screened, including 51 monomicrobial Gram-negative ESKAPEEc isolates. RAST results at 4, 6, 8, and 16–20 h were compared with standard antimicrobial susceptibility testing (AST) obtained using the BD Phoenix™ system. Categorical agreement (CA) and error frequency were calculated. Multiplex PCR and Sanger sequencing were performed on 15 CRKP isolates to identify carbapenemase genes and allelic variants. Results: 51 Gram-negative ESKAPEEc isolates met the inclusion criteria for RAST (15 E. coli, 19 K. pneumoniae, 11 A. baumannii, and 6 P. aeruginosa). Overall performance varied markedly by species and antibiotic. E. coli showed frequent unreadable or ATU zones at early timepoints and wide CA variability (50–100%), with high very major error (VME) rates for AMP, TZP, and CAZ, particularly at 6–8 h. K. pneumoniae displayed consistently high CA (mostly 100%) for carbapenems, CAZ, and TZP. A. baumannii demonstrated excellent agreement (100% for most agents), except for GEN at 6–8 h. P. aeruginosa could be evaluated only at 16–20 h, showing high CA for AMK, CAZ, and CIP; lower CA for MEM (83%); non-calculable CA for IMI due to universal ATU readings; and a CA value of 0% for TZP due to the predominance of the ATU results. VMEs ranged from 0% to 26.1% across species and reading times, but carbapenems did not generate VMEs. Molecular analysis revealed bla_KPC in 66.7%, bla_NDM in 46.7%, and bla_OXA-48 in 33.3% of isolates, with co-occurrence in several strains. Sequencing identified bla_KPC-2 and bla_NDM-1 as the predominant variants, with one isolate harboring bla_NDM-5. Conclusions: EUCAST RAST markedly accelerates susceptibility reporting from positive blood cultures, but its accuracy is species- and time-dependent. Performance was excellent for K. pneumoniae (including CRKP) and A. baumannii and acceptable for P. aeruginosa at 16–20 h. In contrast, E. coli showed frequent ATU results at early timepoints and high ME/VME rates, making readings before 8 h unreliable for clinical decisions. Overall, RAST can effectively support rapid antimicrobial stewardship when species-specific limitations are recognized, and early-timepoint results are interpreted with caution. Full article