Clinical impact of time to results from the microbiology laboratory in bloodstream infections caused by carbapenemase-producing Enterobacterales (TIME-CPE STUDY).

OBJECTIVES: To evaluate the impact of time to results (TTR) on the outcome of patients with carbapenemase-producing Enterobacterales bloodstream infections (CPE-BSI). METHODS: Times-series study conducted from January 2014 to December 2021, selecting patients with first CPE-BSI episodes. Periods of intervention were defined according to implementation of diagnostic bundle tests in the microbiology laboratory: pre-intervention (January 2014-December 2017) and post-intervention (January 2018-December 2021). TTR was defined as time elapsed from positivity time of the blood culture bottles to physicians' notification of CPE-BSI episodes, and was evaluated in patients who received inappropriate empirical and switched to appropriate targeted treatment (switch group). Analysis of a composite unfavourable outcome (mortality at Day 30 and/or persistent and/or recurrent bacteraemia) was performed for the total episodes and in the switch group. RESULTS: One hundred and nine episodes were analysed: 66 pre-intervention and 43 post-intervention. Compared with pre-intervention, patients in the post-intervention period were younger (68 versus 63 years, P = 0.04), had INCREMENT score > 7 (31.8% versus 53.5%, P = 0.02) and unfavourable outcome (37.9% versus 20.9%, P = 0.04). Proportion of TTR > 30 h was more frequent pre-intervention than post-intervention (61.7% versus 35.5%, P = 0.02). In multivariate analysis of the 109 episodes, source other than urinary or biliary (OR 2.76, 95% CI 1.11-6.86) was associated with unfavourable outcome, while targeted appropriate treatment trended to being protective (OR 0.17, 95% CI 0.03-1.00). Considering the switch group (n = 78), source other than urinary or biliary (OR 14.9, 95% CI 3.25-69.05) and TTR > 30 h (OR 4.72, 95% CI 1.29-17.22) were associated with unfavourable outcome. CONCLUSIONS: Decreased TTR in the post-intervention period was associated with the outcome in patients with CPE-BSI episodes.

Significant increase of CTX-M-15-ST131 and emergence of CTX-M-27-ST131 Escherichia coli high-risk clones causing healthcare-associated bacteraemia of urinary origin in Spain (ITUBRAS-2 project).

OBJECTIVES: To assess the microbiological characteristics of Escherichia coli causing healthcare-associated bacteraemia of urinary origin (HCA-BUO) in Spain (ITUBRAS-2 project), with particular focus on ESBL producers and isolates belonging to ST131 high-risk clone (HiRC). Clinical characteristics and outcomes associated with ST131 infection were investigated. METHODS: A total of 222 E. coli blood isolates were prospectively collected from patients with HCA-BUO from 12 tertiary-care hospitals in Spain (2017-19). Antimicrobial susceptibility and ESBL/carbapenemase production were determined. ST131 subtyping was performed. A subset of 115 isolates were selected for WGS to determine population structure, resistome and virulome. Clinical charts were reviewed. RESULTS: ESBL-producing E. coli prevalence was 30.6% (68/222). ST131 represented 29.7% (66/222) of E. coli isolates and accounted for the majority of ESBL producers (46/68, 67.6%). The C2/H30-Rx subclone accounted for most ST131 isolates (44/66) and was associated with CTX-M-15 (37/44) and OXA-1 enzymes (27/44). Cluster C1-M27 was identified in 4/10 isolates belonging to subclade C1/H30-R1 and associated with CTX-M-27. Additionally, ST131 isolates showed a high content of other acquired resistance genes, and clade C/ST131 isolates carried characteristic QRDR mutations. They were categorized as uropathogenic E. coli and had higher aggregate virulence scores. ST131 infection was associated with more complex patients, prior use of cephalosporins and inadequate empirical treatment but was not associated with worse clinical outcomes. CONCLUSIONS: ST131 HiRC is the main driver of ESBL-producing E. coli causing HCA-BUO in Spain, mainly associated with the expansion of subclade CTX-M-15-C2/H30-Rx and the emergence of CTX-M-27-C1/H30-R1 (Cluster C1-M27).

Yeasts from blood cultures in the wake of the COVID-19 pandemic in a tertiary care hospital: Shift in species epidemiology, steady low antifungal resistance and full in vitro ibrexafungerp activity.

Several institutions reported a rise not only in fungemia incidence but also in the number of cases caused by Candida auris or fluconazole-resistant C. parapsilosis during the COVID-19 pandemic. Since the pandemic broke out in early 2020, we studied its impact on fungemia incidence, species epidemiology, potential patient-to-patient transmission, and antifungal resistance in 166 incident yeast isolates collected from January 2020 to December 2022. Isolates were molecularly identified, and their antifungal susceptibilities to amphotericin B, azoles, micafungin, anidulafungin, and ibrexafungerp were studied following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) method, and genotyped. The fungemia incidence (episodes per 1000 admissions) tended to decrease over time (2020 = 1.60, 2021 = 1.36, 2022 = 1.16); P > .05). Species distribution was C. albicans (50.6%, n = 84), C. parapsilosis (18.7%, n = 31), C. glabrata (12.0%, n = 20), C. tropicalis (11.4%, n = 19), C. krusei (3.0%, n = 5), other Candida spp. (1.2%, n = 2), and non-Candida yeasts (3.0%, n = 5). The highest and lowest proportions of C. albicans and C. parapsilosis were detected in 2020. The proportion of isolates between 2020 and 2022 decreased in C. albicans (60.3% vs. 36.7%) and increased in C. parapsilosis (10.3% vs. 28.6%; P < .05) and C. tropicalis (8.8% vs. 16.3%; P > .05). Only three C. albicans intra-ward clusters involving two patients each were detected, and the percentages of patients involved in intra-ward clusters reached 9.8% and 8.0% in 2020 and 2021, respectively, suggesting that clonal spreading was not uncontrolled. Fluconazole resistance (5%) exhibited a decreasing trend (P > .05) over time (2020 = 7.6%; 2021 = 4.2%; and 2022 = 2.1%). Ibrexafungerp showed high in vitro activity.

Fungemia incidence increased during the COVID-19 pandemic in our hospital, however, clonal spreading was not uncontrolled. The proportion of C. parapsilosis and C. tropicalis cases constantly increased. Antifungal resistance remained very low, and fluconazole-resistant C. parapsilosis was undetected.

Evidence of Fluconazole-Resistant Candida parapsilosis Genotypes Spreading across Hospitals Located in Madrid, Spain and Harboring the Y132F ERG11p Substitution.

We have been monitoring the antifungal resistance in Candida parapsilosis isolates collected from inpatients at Madrid metropolitan area hospitals for the last 3 years. The study aimed to elucidate the presence of fluconazole-resistant C. parapsilosis genotypes in Madrid. From January 2019 to December 2021, a total of 354 C. parapsilosis isolates (n = 346 patients) from blood (76.6%) or intraabdominal samples were collected and genotyped using species-specific microsatellite markers. Antifungal susceptibilities to amphotericin B, the triazoles, micafungin, anidulafungin, and ibrexafungerp were performed according to EUCAST E.Def 7.3.2; the ERG11 gene was sequenced in fluconazole-resistant isolates. A total of 13.6% (n = 48/354) isolates (one per patient) were found to be resistant to fluconazole and non-wild-type to voriconazole but fully susceptible to ibrexafungerp. Resistant isolates were mostly sourced from blood (n = 45/48, 93.8%) and were detected in five hospitals. Two hospitals accounted for a high proportion of resistant isolates (n = 41/48). Resistant isolates harbored either the Y132F ERG11p amino acid substitution (n = 43) or the G458S substitution (n = 5). Isolates harboring the Y132F substitution clustered into a clonal complex involving three genotypes (one genotype accounted for n = 39/43 isolates) that were found in four hospitals. Isolates harboring the G458S substitution clustered into another genotype found in a fifth hospital. C. parapsilosis genotypes demonstrating resistance to fluconazole have been spreading across hospitals in Madrid, Spain. Over the last 3 years, the frequency of isolation of such isolates and the number of hospitals affected is on the rise.

Blood and intra-abdominal Candida spp. from a multicentre study conducted in Madrid using EUCAST: emergence of fluconazole resistance in Candida parapsilosis, low echinocandin resistance and absence of Candida auris.

OBJECTIVES: We prospectively monitored the epidemiology and antifungal susceptibility of Candida spp. from blood cultures and intra-abdominal samples in patients admitted to hospitals in the Madrid area. METHODS: Between 2019 and 2021, we prospectively collected incident isolates [one per species, patient and compartment (blood cultures versus intra-abdominal samples)] from patients admitted to any of 16 hospitals located in Madrid. We studied the antifungal susceptibilities to amphotericin B, triazoles, micafungin, anidulafungin and ibrexafungerp following the EUCAST E.Def 7.3.2 procedure. RESULTS: A total of 2107 Candida spp. isolates (1895 patients) from blood cultures (51.7%) and intra-abdominal samples were collected. Candida albicans, the Candida glabrata complex, the Candida parapsilosis complex, Candida tropicalis and Candida krusei accounted for 96.9% of the isolates; in contrast, Candida auris was undetected. Fluconazole resistance in Candida spp. was higher in blood cultures than in intra-abdominal samples (9.1% versus 8.2%; P > 0.05), especially for the C. parapsilosis complex (16.6% versus 3.6%, P < 0.05), whereas echinocandin resistance tended to be lower in blood cultures (0.5% versus 1.0%; P > 0.05). Resistance rates have risen, particularly for fluconazole in blood culture isolates, which increased sharply in 2021. Ibrexafungerp showed in vitro activity against most isolates. Species distributions and resistance rates varied among hospitals. CONCLUSIONS: Whereas no C. auris isolates were detected, fluconazole-resistant C. parapsilosis isolates have been spreading across the region and this has pulled up the rate of fluconazole resistance. In contrast, the rate of echinocandin resistance continues to be low.

Prevalence and clinical features of secondary skin lesions in septic patients with bloodstream infections.

Cutaneous manifestations developed in the course of sepsis are poorly documented in the medical literature beyond those related to specific pathogens or classical clinical pictures such as purpura fulminans or ecthyma gangrenosum. The objective of this study was to determine the overall prevalence of sepsis-related skin findings and evaluate their possible impact on the prognosis of septic patients. Single-centre, retrospective study of septic patients with documented bloodstream infections admitted in a tertiary hospital during 2019. Primary skin and soft tissue infections, and non-sepsis-related skin conditions diagnosed during hospital admission were excluded. Unselected sample of 320 episodes of sepsis in 265 patients. Secondary skin lesions were documented in 57 sepsis episodes (17.8%) in 47 patients. Purpura (petechiae/ecchymosis) was the most frequent cutaneous finding in septic patients (35.5%), with non-acral involvement in more than one-third of the episodes (38.5%), followed by skin and soft tissue erythema/oedema (25.8%) and maculopapular rashes (11.3%). Secondary skin lesions occurred more frequently in sepsis of respiratory (p = 0.027) and skin and soft tissue (p = 0.018) origin, as well as in sepsis caused by Pseudomonas aeruginosa and Stenotrophomonas maltophilia (p = 0.001). Mean hospital stay was 38.58 days and sepsis-related mortality 21.1%. Our results suggest that cutaneous involvement in the course of sepsis is frequent, with purpura being the main clinical sign. The semiology described in this study, easily identifiable by non-dermatologists, should alert clinicians to the potential unfavourable course of these patients.

Monitoring the Epidemiology and Antifungal Resistance of Yeasts Causing Fungemia in a Tertiary Care Hospital in Madrid, Spain: Any Relevant Changes in the Last 13 Years?

We conducted an updated analysis on yeast isolates causing fungemia in patients admitted to a tertiary hospital in Madrid, Spain, over a 13-year period. We studied 896 isolates associated with 872 episodes of fungemia in 857 hospitalized patients between January 2007 and December 2019. Antifungal susceptibility was assessed by EUCAST EDef 7.3.2. Mutations conferring azole and echinocandin resistance were further studied, and genotyping of resistant clones was performed with species-specific microsatellite markers. Candida albicans (45.8%) was the most frequently identified species, followed by the Candida parapsilosis complex (26.4%), Candida glabrata (12.3%), Candida tropicalis (7.3%), Candida krusei (2.3%), other Candida spp. (3.1%), and non-Candida yeasts (2.8%). The rate of fluconazole resistance in Candida spp. was 4.7%, ranging from 0% (C. parapsilosis) to 9.1% (C. glabrata). The overall rate of echinocandin resistance was 3.1%. Resistance was highly influenced by the presence of intrinsically resistant species. Although the number of isolates between 2007 and 2013 was almost 2-fold higher than that in the period from 2014 to 2019 (566 versus 330), fluconazole resistance in Candida spp. was greater in the second period (3.5% versus 6.8%; P < 0.05), while overall resistance to echinocandins remained stable (3.5% versus 2.4%; P > 0.05). Resistant clones were collected from different wards and/or time points, suggesting that there were no epidemiological links. The number of fungemia episodes has been decreasing over the last 13 years, with a slight increase in the rate of fluconazole resistance and stable echinocandin resistance. Antifungal resistance is not the cause of the spread of resistant clones.

Antifungal Susceptibility Testing Identifies the Abdominal Cavity as a Source of Candida glabrata-Resistant Isolates.

To identify unrecognized niches of resistant Candida isolates and compartmentalization, we retrospectively studied the antifungal susceptibility of 1,103 Candida spp. isolates from blood cultures, nonblood sterile samples, and nonsterile samples. Antifungal susceptibility was assessed by EUCAST E.Def 7.3.2; sequencing and genotyping of the fks1-2 and erg11 genes were carried out for non-wild-type isolates. Resistance compartmentalization (presence of resistant and susceptible isogenic isolates in different anatomical sites of a given patient) was studied. Clinical charts of patients carrying non-wild-type isolates were reviewed. Most isolates (63%) were Candida albicans, regardless the clinical source; Candida glabrata (27%) was the second most frequently found species in abdominal cavity samples. Fluconazole and echinocandin resistance rates were 1.5 and 1.3%, respectively, and were highest in C. glabrata. We found 22 genotypes among non-wild-type isolates, none of them widespread across the hospital. Fluconazole/echinocandin resistance rates of isolates from the abdominal cavity (3.2%/3.2%) tended to be higher than those from blood cultures (0.7%/1.3%). Overall, 15 patients with different forms of candidiasis were infected by resistant isolates, 80% of whom had received antifungals before or at the time of isolate collection; resistance compartmentalization was found in six patients, mainly due to C. glabrata. The highest antifungal resistance rate was detected in isolates from the abdominal cavity, mostly C. glabrata. Resistance was not caused by the spread of resistant clones but because of antifungal treatment. Resistance compartmentalization illustrates how resistance might be overlooked if susceptibility testing is restricted to bloodstream isolates.

Whole genome sequencing confirms Candida albicans and Candida parapsilosis microsatellite sporadic and persistent clones causing outbreaks of candidemia in neonates.

Whole genome sequencing has been extensively used to describe infection outbreaks, although with limited application on Candida albicans and Candida parapsilosis.We retrospectively studied all patients admitted to the neonatal care unit diagnosed with candidemia caused by C. albicans (n = 46) or C. parapsilosis (n = 31) between 2007 and 2010 (Period 1) and 2011 and 2014 (Period 2). All isolates were genotyped by microsatellite markers. A cluster was defined as a group of ≥ 2 patients infected by strains with identical genotypes. For the validation of microsatellite markers and outbreak investigation, phylogenetic analyses and whole genome pairwise strain comparisons were performed.The number of episodes was significantly higher in Period 1 than in Period 2 (51 vs 32; P = 0.003); the reduction in the number of cases coincided with the educational campaign for catheter care implementation in 2011. Overall, eight genotypes were clusters involving 29 patients. All C. albicans (n = 5) and C. parapsilosis (n = 3) clusters were found during Period 1 before the educational campaign. No statistically significant differences were found between the percentage of C. albicans and C. parapsilosis clusters, but the percentage of patients associated to the clusters was significantly higher for C. parapsilosis clusters in comparison to C. albicans clusters (52 vs 28.2%; P = 0.03). Whole genome sequencing confirmed microsatellite-defined clusters with high bootstrap values.Whole genome sequences confirmed microsatellite-defined clusters, corroborating the presence of outbreaks. Persistent or sporadic Candida clusters causing candidemia in neonates disappeared after the implementation of catheter care educational campaigns. LAY SUMMARY: We retrospectively studied all patients admitted to the neonatal care unit diagnosed with candidemia caused by C. albicans or C. parapsilosis. Reliable whole genome sequences confirmed microsatellite-defined clusters, corroborating the presence of outbreaks before educational campaigns for catheter care.

Clinical Relevance and Prognostic Value of Persistently Negative (1,3)-ß-D-Glucan in Adults With Candidemia: A 5-year Experience in a Tertiary Hospital.

BACKGROUND: The clinical relevance and the potential prognostic role of persistently negative (1,3)-ß-D-glucan (BDG) in adults with proven candidemia is unknown. METHODS: This retrospective study included all adults diagnosed with candidemia our tertiary university hospital from 2012-2017 who had at least 2 serum BDG determinations throughout the episode of fungemia (Fungitell Assay; positive cut-off ≥80pg/mL). Epidemiology and clinical outcomes were compared between patients with all negative versus any positive BDG tests. Poor clinical outcomes included complications due to candidemia or 30-day all-cause mortality. RESULTS: Overall, 26/148 (17.6%) candidemic adults had persistently negative BDG tests. These patients were less likely to present Candida growth in all 3 sets of blood cultures (15.4% vs 45.1%; P = .005) and had less severe clinical presentations (median Pitt score, 0 [interquartile range {IQR} 0-1] vs 1 [IQR 0-2] in patients with any positive BDG test; P = .039). Although adequate treatment was equally provided to both groups (96.2% in persistently negative group vs 93.4 in positive group; P = .599), the persistently negative group had a higher rate of microbiological clearance in the first follow-up blood cultures (92.3% vs 69.7% in positive group; P = .005), fewer complications due to candidemia (7.7% vs 33.6% in positive group; P = .008), a lower 30-day mortality rate (3.8% vs 23.8% in positive group; P = .004), and a shorter in-hospital stay (34 days [IQR 18-55] vs 51 days [IQR 35-91] in positive group; P = .003). In the multivariate analysis, persistently negative BDG tests were independently associated with better prognoses (odds ratio 0.12, 95% confidence interval 0.03-0.49; P = .003). CONCLUSIONS: Candidemic patients with persistently negative BDG tests present a better prognosis than the comparative group, probably due to a lower systemic fungal burden. In this context, the appropriate use of persistently negative BDG results could be an aid to individualize therapeutic management in the near future.

Susceptibility of uncommon Candida species to systemic antifungals by the EUCAST methodology.

The incidence of infections by uncommon Candida species has increased in recent years, however, in vitro susceptibility data are scarce. Here we assess the susceptibility of C. krusei, C. dubliniensis, C. lusitaniae, and C. guilliermondii complex isolates (n = 120) to antifungal agents by the EUCAST methodology. C. dubliniensis proved to be the most susceptible species, similar to that of C. albicans (P < .05), whereas C. guilliermondii was the least susceptible. Two C. krusei isolates were echinocandin-resistant and harbored a point mutation (L701M) in the FKS1. Some isolates were either fluconazole-resistant (C. lusitaniae, n = 2) or fluconazole non-wild type (C. guilliermondii, n = 3).

A retrospective cohort of invasive fusariosis in the era of antimould prophylaxis.

Mould-active prophylaxis is affecting the epidemiology of invasive mycoses in the form of a shift toward less common entities such as fusariosis. We analyze the characteristics of invasive fusariosis and its association to antifungal prophylaxis in a retrospective cohort (2004-2017) from a tertiary hospital in Madrid, Spain. Epidemiological, clinical, microbiological, and antifungal consumption data were retrieved. Isolates were identified to molecular level, and antifungal susceptibility was tested. Eight cases of invasive fusariosis were diagnosed. Three periods were identified according to incidence: <2008 (three cases), 2008-2013 (zero cases), >2014 (five cases). All except one case involved breakthrough fusariosis. During the earliest period, the episodes occurred while the patient was taking itraconazole (two) or fluconazole (one); more recently, while on micafungin (three) or posaconazole (one). Early cases involved acute leukemia at induction/consolidation, recent cases relapsed/refractory disease (P = .029). Main risk factor for fusariosis (62.5%) was prolonged neutropenia (median 44 days). Galactomannan and beta-D-glucan were positive in 37.5% and 100% of cases, respectively. All isolates except F. proliferatum presented high minimal inhibitory concentrations (MICs) against the azoles and lower MIC to amphotericin B. Most patients received combined therapy. Mortality at 42 days was 62.5%. Resolution of neutropenia was associated with survival (P = .048). Invasive fusariosis occurs as breakthrough infection in patients with hematologic malignancy, prolonged neutropenia, and positive fungal biomarkers. Recent cases were diagnosed in a period of predominant micafungin use in patients who had more advanced disease and protracted neutropenia and for whom mortality was extremely high. Resolution of neutropenia was a favorable prognostic factor.

Candidemia Candida albicans clusters have higher tendency to form biofilms than singleton genotypes†.

The capacity of Candida spp. to form biofilms allows them to attach either to living or inert surfaces, promoting their persistence in hospital environments. In a previous study, we reported strain-to-strain variations in Candida spp. biofilm development, suggesting that some genotypes may be greater biofilm formers than others. In this study, we hypothesize that isolates pertaining to clusters may be found more frequently in the environment due to their ability to form biofilms compared to singleton genotypes. Two hundred and thirty-nine Candida spp. isolates (78 clusters) from candidemia patients admitted to 16 hospitals located in different cities and countries-and the same number of singleton genotypes used as controls-were tested in terms of biofilm formation using the crystal violet and the XTT reduction assays. Candida albicans clusters showed higher biofilm formation in comparison to singleton genotypes (P < .01). The biofilms formed by intra-hospital C. albicans clusters showed higher metabolic activity (P < .05). Furthermore, marked variability was found among species and type of cluster. We observed that the higher the number of isolates, the higher the variability of biofilm production by isolates within the cluster, suggesting that the production of biofilm by isolates of the same genotype is quite diverse and does not depend on the type of cluster studied. In conclusion, candidemia Candida spp. clusters-particularly in the case of C. albicans-show significantly more biomass production and metabolic activity than singleton genotypes.

Detection of Echinocandin-Resistant Candida glabrata in Blood Cultures Spiked with Different Percentages of FKS2 Mutants.

Infections caused by the coexistence of Candida glabrata echinocandin-resistant and echinocandin-susceptible cells may be possible, and the detection of FKS mutants when the proportions of FKS mutants are underrepresented poses a problem. We assessed the role of EUCAST and methods directly performed on positive blood cultures-Etest (ETDIR) and anidulafungin-containing agar plate assays-for detecting resistance in C. glabrata isolates containing different amounts of echinocandin-susceptible and -resistant Candida glabrata isolates. We studied 10 pairs of C. glabrata isolates involving parental echinocandin-susceptible isolates and isogenic echinocandin-resistant FKS mutant isolates. Three inocula per pair (1 × 103 to 5 × 103, 1 × 102 to 5 × 102, and 10 to 50 CFU/ml) spanning suspensions with different amounts of susceptible/resistant isolates (9/1, 5/5, and 1/9 proportions for each the three inocula) were prepared. The suspensions were spiked in Bactec bottles and incubated until they were positive, and the three methods were compared. The EUCAST method showed echinocandin resistance when the bottles were spiked with susceptible/resistant isolates at 5/5 and 1/9 proportions; the results for the suspensions with a 9/1 proportion of susceptible/resistant isolates were susceptible for three pairs. We observed with the ETDIR resistance to both echinocandins in all pairs (resistance to micafungin and anidulafungin; MICs, ≥0.064 mg/liter and ≥0.125 mg/liter, respectively) and a double ring of growth inhibition in two pairs. The anidulafungin-containing plates showed fungal growth in the 90 spiked blood cultures at 48 h. Testing of echinocandin susceptibility with the ETDIR directly on the positive blood culture bottles is a reliable and rapid method to detect echinocandin resistance in C. glabrata On the other hand, resistance can be missed with the EUCAST method when resistant isolates are underrepresented.

MSH2 Gene Point Mutations Are Not Antifungal Resistance Markers in Candida glabrata.

The high rates of antifungal resistance in Candida glabrata may be facilitated by the presence of alterations in the MSH2 gene. We aimed to study the sequence of the MSH2 gene in 124 invasive C. glabrata isolates causing incident episodes of candidemia (n = 81), subsequent candidemia episodes (n = 9), endocarditis (n = 2), and in vitro-generated echinocandin-resistant isolates (n = 32) and assessed its relationship with genotypes, acquisition of antifungal resistance in vivo and in vitro, and patient prognosis. The MSH2 gene was sequenced, and isolates were genotyped using six microsatellite markers and multilocus sequence typing (MLST) based on six housekeeping genes. According to EUCAST, isolates causing candidemia (n = 90) were echinocandin susceptible, and four of them were fluconazole resistant (MIC ≥64 mg/liter). One isolate obtained from a heart valve was resistant to micafungin and anidulafungin (MICs, 2 mg/liter and 1 mg/liter, respectively). MSH2 gene mutations were present in 44.4% of the incident isolates, the most common being V239L. The presence of MSH2 mutations was not correlated with in vitro or in vivo antifungal resistance. Microsatellite and MLST revealed 27 genotypes and 17 sequence types, respectively. Fluconazole-resistant isolates were unrelated. Most MSH2 mutations were found in cluster isolates; conversely, some mutations were found in more than one genotype. No clinical differences, including previous antifungal use, were found between patients infected by wild-type MSH2 gene isolates and isolates with any point mutation. The presence of MSH2 gene mutations in C. glabrata isolates causing candidemia is not correlated with specific genotypes, the promotion of antifungal resistance, or the clinical outcome.

Evaluation of the Alfred AST® system for rapid antimicrobial susceptibility testing directly from positive blood cultures.

To assess the concordance of antimicrobial susceptibility testing results obtained by the Alfred AST® system performed directly from positive blood cultures in comparison with the standard susceptibility test results performed from isolated colonies by an automated broth microdilution method and to determine the applicability of Alfred AST® system in the routine of our blood culture laboratory. This system is based on the detection of growth by turbidimetry through a technology based on light scattering. Antimicrobial susceptibility testing was performed directly from positive bottles by the Alfred AST® system (Alifax, Padova, Italy). The broth microdilution method (MicroScan, Beckman Coulter, CA, USA) performed to the isolates was considered the standard for comparison. We evaluated 115 significant episodes of bacteremia produced by 51 Gram-negative Enterobacterales, 8 Pseudomonas spp., 2 non-fermenting Gram-negative rods, 7 Staphylococcus aureus, 23 coagulase-negative Staphylococcus, 12 Enterococcus spp., and 12 Streptococcus spp. We performed 828 susceptibility determinations with a categorical agreement with the standard method of 97.1%. Only 24 errors (2.9%) were detected. It should be pointed out that for staphylococci and glycopeptides the correlation was only 87% and for non-fermenting Gram-negative rods and piperacillin/tazobactam was only 88.9%. Time to get antibiogram results by Alfred AST® system was 5 versus 48 h for the standard microdilution method from the isolated colonies. The Alfred AST® system is a useful and rapid method to obtain antimicrobial susceptibility results within the same work shift after blood culture positivity.

Evaluation of the Alfred™ turbidity monitoring system (Alifax®) following sonication in the diagnosis of central venous catheter colonization.

The conventional diagnostic techniques for catheter colonization (CC) take at least 48 h to yield results. Therefore, new diagnostic procedures that speed up the time necessary for results are needed. Our main objective was to assess the efficacy of the combination of sonication, turbidity monitoring, and MALDI-TOF to detect CC and catheter-related bloodstream infection (C-RBSI). For 1 year, we assessed central venous catheter (CVC) tips that arrived at the microbiology laboratory from adult patients admitted to our institution. CVC tips were cut, inoculated into 2.5 ml of BHI, and sonicated for 1 min. The suspension was then processed using Gram stain, quantitative culture (gold standard), and preincubation on the Alfred™ system. We analyzed the validity values of our new diagnostic approach for prediction of CC and C-RBSI and compared them with those of the gold standard. We collected a total of 167 catheters, 33 (19.8%) of which were colonized. We confirmed 21 episodes of C-RBSI. The distribution of microorganisms in colonized CVCs was as follows: Gram-positive, 68.4%; Gram-negative, 5.3%; and yeasts, 26.3%. The validity values for CC and C-RBSI using the new procedure were as follows: S, 39.4%/61.9%; Sp, 100%/100%; PPV, 100%/100%; and NPV, 87.0%/94.8%. The combination of sonication with a pre-incubation period based on turbidity monitoring using the Alfred™ system followed by MALDI-TOF proved to be a useful tool that was faster than conventional culture for ruling out C-RBSI. Future studies are needed to assess the clinical and economic impact of this diagnostic approach.

Growth kinetics in Candida spp.: Differences between species and potential impact on antifungal susceptibility testing as described by the EUCAST.

We studied the growth kinetic parameters of clinically relevant Candida species to verify the differences between species following the incubation and medium conditions recommended by the EUCAST. We analyzed 705 susceptible Candida spp. from patients with candidemia and Candida glabrata isolates resistant to echinocandins or fluconazole (n = 38) and calculated the average growth rate, maximum peak, time to maximum rate, and lag phase. We also examined inter- and intra-species differences, as well as the percentage of isolates reaching an optical density of 0.2 over time. Interspecies differences in growth phases and kinetic parameters were found. C. glabrata was the fastest growing species and the lag phase of C. parapsilosis was longer than that of the other species considered in this study. Strain-to-strain variations were found between species. A positive correlation between the average growth rate and maximum peak was determined. Echinocandin-resistant C. glabrata isolates had significantly lower average growth rate but higher time to maximum rate in comparison to wild-type C. glabrata isolates. Incubation periods of 12-15 hours allowed reaching the 0.2 optical density threshold in 100% of C. glabrata, C. tropicalis, and C. krusei isolates. We show differences in kinetic parameters between Candida spp. C. glabrata was the fastest growing species and C. parapsilosis showed the longest lag phase. Resistance to echinocandins may affect the growth kinetic curve. Speeding up antifungal susceptibility results could be possible for some isolates, particularly C. glabrata, C. tropicalis, and C. krusei.

Resistance to Echinocandins in Candida Can Be Detected by Performing the Etest Directly on Blood Culture Samples.

We examined the rapid evaluation of susceptibility to echinocandins in Candida spp. using the Etest performed directly on positive blood cultures and anidulafungin-containing agar plates. We prospectively collected 80 positive blood cultures (Bactec-FX system, Becton-Dickinson, Cockeysville, MD, USA) with echinocandin-susceptible Candida spp. (n = 60) and echinocandin-intermediate Candida parapsilosis (n = 20) from patients with candidemia. Additionally, blood culture bottles of nonfungemic/bacteremic patients were spiked with 35 echinocandin-resistant Candida species isolates. A total of 2 to 4 drops of medium from each bottle were stroked directly onto both RPMI 1640 agar plates with micafungin and anidulafungin Etest strips (ETDIR) and Sabouraud agar plates containing 2 mg/liter of anidulafungin. The isolates were tested according to the EUCAST method and Etest standard (ETSD). Essential and categorical agreement between the methods was calculated. The essential agreement and categorical agreement between the EUCAST method and ETDIR and ETSD were both >97.4%. The essential agreement between ETDIR and the EUCAST method for both echinocandins was >97%. The categorical agreement between the FKS sequence and ETDIR was 97.4%. The ETDIR MICs of anidulafungin and micafungin (≥0.19 mg/liter and ≥0.064 mg/liter, respectively) effectively separated all susceptible FKS wild-type isolates from the resistant FKS mutant isolates. The categorical agreement (62.6%) between the EUCAST method and growth on anidulafungin-containing plates was poor, with the best agreement observed for Candida glabrata (94.2%). When performed directly on positive blood cultures from patients with candidemia, the Etest with micafungin and anidulafungin is a reliable procedure for the rapid testing of susceptibility to echinocandins in Candida species isolates.

Mutant Prevention Concentration and Mutant Selection Window of Micafungin and Anidulafungin in Clinical Candida glabrata Isolates.

We report the mutant prevention concentration (MPC) and mutant selection window (MSW) for micafungin and anidulafungin administered to treat Candida glabrata We also determine the mutation frequency. We studied 20 echinocandin-susceptible, fluconazole-intermediate, and FKS wild-type C. glabrata isolates. Adjusted inocula were stroked directly onto Sabouraud agar plates containing different concentrations of micafungin or anidulafungin and visually inspected daily for up to 5 days of incubation. Individual colonies growing on the plates containing echinocandins at 1 mg/liter were selected for antifungal susceptibility testing. The FKS genes of the resulting individual phenotypically resistant colonies were sequenced, and the MPC, MSW, and mutation frequency were determined. Biofilm was quantified, and the growth kinetics and virulence (Galleria mellonella model) of the resulting individual FKS mutant colonies were studied. For micafungin and anidulafungin, we found similar results for the MPC (0.06 to 2 mg/liter and 0.25 to 2 mg/liter, respectively), MSW (0.015 to 2 mg/liter for both echinocandins), and mutation frequency (3.7 × 10-8 and 2.8 × 10-8, respectively). A total of 12 isolates were able to grow at 1 mg/liter on echinocandin-containing plates, yielding a total of 32 phenotypically resistant colonies; however, FKS2 mutations (ΔF658, S663P, W715L, and E655A) were observed only in 21 colonies. We did not find differences in biofilm formation, the kinetic parameters studied, or the median survival of larvae infected by wild-type isolates and the resulting individual FKS2 mutant colonies. Echinocandin concentrations lower than 2 mg/liter can lead to selection of resistance mutations in C. glabrata isolates in vitro.