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Background: The rise of multi-drug-resistant Gram-negative bacteria necessitates the development of new antimicrobial agents. Cefiderocol shows promising activity by exploiting bacterial iron transport systems to penetrate the outer membranes of resistant pathogens. Objectives: This study evaluates the efficacy of cefiderocol testing methods and trailing effect impact using a ComASP® Cefiderocol panel, disk diffusion (DD), and MIC test strips (MTS) compared to iron-depleted broth microdilution (ID-BMD). Methods: A total of 131 Gram-negative strains from clinical samples was tested by commercial methods and the gold standard. Results were interpreted as per 2024 and 2023 EUCAST guidelines. Results: ID-BMD revealed high cefiderocol susceptibility among Enterobacterales and Pseudomonas aeruginosa, with one Klebsiella pneumoniae isolate being resistant. Acinetobacter baumannii exhibited higher MIC values, particularly considering trailing effects that complicated MIC readings. ComASP® showed 97% categorical agreement (CA) and 66% essential agreement (EA) with ID-BMD for Enterobacterales but failed to detect the resistant K. pneumoniae. DD tests demonstrated variable CA (72% or 93%), and 38% or 34% of strains within the ATU according to EUCAST Breakpoint Tables v13.0 and 14.0, respectively, with major errors only. MTS for P. aeruginosa had 100% CA but 44% EA, and often underestimated MIC values. Conclusions: The study emphasizes the need for standardized criteria to address trailing effects and ATU and highlights the discrepancies between testing methods. While cefiderocol resistance remains rare, accurate susceptibility testing is crucial for its effective clinical use. The findings suggest that current commercial tests have limitations, necessitating careful interpretation and potential supplementary testing to guide appropriate antibiotic therapy. [ABSTRACT FROM AUTHOR]

Background: The rise of multi-drug-resistant Gram-negative bacteria necessitates the development of new antimicrobial agents. Cefiderocol shows promising activity by exploiting bacterial iron transport systems to penetrate the outer membranes of resistant pathogens. Objectives: This study evaluates the efficacy of cefiderocol testing methods and trailing effect impact using a ComASP® Cefiderocol panel, disk diffusion (DD), and MIC test strips (MTS) compared to iron-depleted broth microdilution (ID-BMD). Methods: A total of 131 Gram-negative strains from clinical samples was tested by commercial methods and the gold standard. Results were interpreted as per 2024 and 2023 EUCAST guidelines. Results: ID-BMD revealed high cefiderocol susceptibility among Enterobacterales and Pseudomonas aeruginosa, with one Klebsiella pneumoniae isolate being resistant. Acinetobacter baumannii exhibited higher MIC values, particularly considering trailing effects that complicated MIC readings. ComASP® showed 97% categorical agreement (CA) and 66% essential agreement (EA) with ID-BMD for Enterobacterales but failed to detect the resistant K. pneumoniae. DD tests demonstrated variable CA (72% or 93%), and 38% or 34% of strains within the ATU according to EUCAST Breakpoint Tables v13.0 and 14.0, respectively, with major errors only. MTS for P. aeruginosa had 100% CA but 44% EA, and often underestimated MIC values. Conclusions: The study emphasizes the need for standardized criteria to address trailing effects and ATU and highlights the discrepancies between testing methods. While cefiderocol resistance remains rare, accurate susceptibility testing is crucial for its effective clinical use. The findings suggest that current commercial tests have limitations, necessitating careful interpretation and potential supplementary testing to guide appropriate antibiotic therapy.

Candida auris has emerged as a fungal pathogen that causes nosocomial outbreaks worldwide. Diseases caused by this fungus are of concern, due to its reduced susceptibility to several antifungals. C. auris exhibits paradoxical growth (PG; defined as growth at high, but not intermediate antifungal concentrations) in the presence of caspofungin (CPF). We have characterized the cellular changes associated with adaptation to CPF. Using EUCAST AFST protocols, all C. auris isolates tested showed PG to CPF, although in some isolates it was more prominent. Most isolates also showed a trailing effect (TE) to micafungin and anidulafungin. We identified two FKS genes in C. auris that encode the echinocandins target, namely β-1,3-glucan synthase. FKS1 contained the consensus hot-spot (HS) 1 and HS2 sequences. FKS2 only contained the HS1 region which had a change (F635Y), that has been shown to confer resistance to echinocandins in C. glabrata. PG has been characterized in other species, mainly C. albicans, where high CPF concentrations induced an increase in chitin, cell volume and aggregation. In C. auris CPF only induced a slight accumulation of chitin, and none of the other phenomena. RNAseq experiments demonstrated that CPF induced the expression of genes encoding several GPI-anchored cell wall proteins, membrane proteins required for the stability of the cell wall, chitin synthase and mitogen-activated protein kinases (MAPKs) involved in cell integrity, such as BCK2, HOG1 and MKC1 (SLT2). Our work highlights some of the processes induced in C. auris to adapt to echinocandins.

Candida auris has emerged as a fungal pathogen that causes nosocomial outbreaks worldwide. Diseases caused by this fungus are of concern, due to its reduced susceptibility to several antifungals. C. auris exhibits paradoxical growth (PG; defined as growth at high, but not intermediate antifungal concentrations) in the presence of caspofungin (CPF). We have characterized the cellular changes associated with adaptation to CPF. Using EUCAST AFST protocols, all C. auris isolates tested showed PG to CPF, although in some isolates it was more prominent. Most isolates also showed a trailing effect (TE) to micafungin and anidulafungin. We identified two FKS genes in C. auris that encode the echinocandins target, namely β-1,3-glucan synthase. FKS1 contained the consensus hot-spot (HS) 1 and HS2 sequences. FKS2 only contained the HS1 region which had a change (F635Y), that has been shown to confer resistance to echinocandins in C. glabrata. PG has been characterized in other species, mainly C. albicans, where high CPF concentrations induced an increase in chitin, cell volume and aggregation. In C. auris CPF only induced a slight accumulation of chitin, and none of the other phenomena. RNAseq experiments demonstrated that CPF induced the expression of genes encoding several GPI-anchored cell wall proteins, membrane proteins required for the stability of the cell wall, chitin synthase and mitogen-activated protein kinases (MAPKs) involved in cell integrity, such as BCK2, HOG1 and MKC1 (SLT2). Our work highlights some of the processes induced in C. auris to adapt to echinocandins. [ABSTRACT FROM AUTHOR]

Purpose: To investigate (i) the dosimetric leaf gap (DLG) and the effect of the "trailing distance" between leaves from different multileaf collimator (MLC) layers in Halcyon systems and (ii) the ability of the currently available treatment planning systems (TPSs) to approximate this effect. Methods: DICOM plans with transmission beams and sweeping gap tests were created in Python for measuring the DLG for each MLC layer independently and for both layers combined. In clinical Halcyon plans both MLC layers are interchangeably used and leaves from different layers are offset, thus forming a trailing pattern. To characterize the impact of such configuration, new tests called "trailing sweeping gaps" were designed and created where the leaves from one layer follow the leaves from the other layer at a fixed "trailing distance" t between the tips. Measurements were carried out on five Halcyons SX2 from different institutions and calculations from both the Eclipse and RayStation TPSs were compared with measurements. Results: The dose accumulated during a sweeping gap delivery progressively increased with the trailing distance t. We call this "the trailing effect." It is most pronounced for t between 0 and 5 mm, although some changes were obtained up to 20 mm. The dose variation was independent of the gap size. The measured DLG values also increased with t up to 20 mm, again with the steepest variation between 0 and 5 mm. Measured DLG values were negative at t = 0 (the leaves from both layers at the same position) but changed sign for t ≥ 1 mm, in line with the positive DLG sign usually observed with single‐layer rounded‐end MLCs. The Eclipse TPS does not explicitly model the leaf tip and, as a consequence, could not predict the dose reduction due to the trailing effect. This resulted in dose discrepancies up to +10% and −8% for the 5 mm sweeping gap and up to ±5% for the 10 mm one depending on the distance t. RayStation implements a simple model of the leaf tip that was able to approximate the trailing effect and improved the agreement with measured doses. In particular, with a prototype version of RayStation that assigned a higher transmission at the leaf tip the agreement with measured doses was within ±3% even for the 5 mm gap. The five Halcyon systems behaved very similarly but differences in the DLG around 0.2 mm were found across different treatment units and between MLC layers from the same system. The DLG for the proximal layer was consistently higher than for the distal layer, with differences ranging between 0.10 mm and 0.24 mm. Conclusions: The trailing distance between the leaves from different layers substantially affected the doses delivered by sweeping gaps and the measured DLG values. Stacked MLCs introduce a new level of complexity in TPSs, which ideally need to implement an explicit model of the leaf tip in order to reproduce the trailing effect. Dynamic tests called "trailing sweeping gaps" were designed that are useful for characterizing and commissioning dual‐layer MLC systems. [ABSTRACT FROM AUTHOR]

Rezafungin is a next-generation echinocandin that has favorable pharmacokinetic properties. We compared the occurrence of paradoxical growth (PG) and trailing effect (TE) characteristics to echinocadins with rezafungin, caspofungin, micafungin and anidulafungin using 365 clinical Candida isolates belonging to 13 species. MICs were determined by BMD method according to CLSI (M27 Ed4). Disconnected growth (PG plus TE) was most frequent with caspofungin (49.6%), followed by anidulafungin (33.7%), micafungin (25.7%), while it was least frequent with rezafungin (16.9%). PG was relatively common in the case of caspofungin (30.1%) but was rare in the case of rezafungin (3.0%). C. tropicalis, C. albicans, C. orthopsilosis and C. inconspicua exhibited PG most frequently with caspofungin, micafungin or anidulafungin. PG never occurred in the case of C. krusei isolates. Against C. tropicalis and C. albicans, echinocandins frequently showed PG after 24 h followed by TE after 48 h. All four echinocandins exhibited TE for the majority of C. auris and C. dubliniensis isolates. Disconnected growth was common among Candida species and was echinocandin- and species-dependent. In contrast to earlier echinocandins, PG was infrequently found with rezafungin. [ABSTRACT FROM AUTHOR]

Rezafungin is a next-generation echinocandin that has favorable pharmacokinetic properties. We compared the occurrence of paradoxical growth (PG) and trailing effect (TE) characteristics to echinocadins with rezafungin, caspofungin, micafungin and anidulafungin using 365 clinical Candida isolates belonging to 13 species. MICs were determined by BMD method according to CLSI (M27 Ed4). Disconnected growth (PG plus TE) was most frequent with caspofungin (49.6%), followed by anidulafungin (33.7%), micafungin (25.7%), while it was least frequent with rezafungin (16.9%). PG was relatively common in the case of caspofungin (30.1%) but was rare in the case of rezafungin (3.0%). C. tropicalis, C. albicans, C. orthopsilosis and C. inconspicua exhibited PG most frequently with caspofungin, micafungin or anidulafungin. PG never occurred in the case of C. krusei isolates. Against C. tropicalis and C. albicans, echinocandins frequently showed PG after 24 h followed by TE after 48 h. All four echinocandins exhibited TE for the majority of C. auris and C. dubliniensis isolates. Disconnected growth was common among Candida species and was echinocandin- and species-dependent. In contrast to earlier echinocandins, PG was infrequently found with rezafungin.

Minimum inhibitory concentration (MIC) of slow growing mycobacteria (SGM) often do not correlate with the treatment response. Among the challenges is the identification of MIC of drugs that degrade in solution faster than the doubling time of the SGM. First, we identified the rate of omadacycline degradation in solution, and its effect on the rapidly growing methicillin resistant Staphylococcus aureus (MRSA). We then identified doubling times versus MICs for Mycobacterium abscessus , M. avium , and M. kansasii , with and without supplementation for degraded drug. Omadacycline concentration in solution declined ∼50% over 24hr. In the MRSA experiments, omadacycline demonstrated 66.48 ± 19.38% loss in potency over 24hr, confirming the degradation rate in solution. M. abscessus had a doubling time of 8.75 ± 1.23hr and the omadacycline MIC after 24hr of incubation was 2mg/L with and without 50% daily drug supplementation, indicating that drug degradation had no effect on this rapid grower. The doubling time for M. avium was 29.52hr (95% confidence interval (CI): 23.18–33.89hr) and 31.15hr (95%CI: 19.45–38.49 hr) for M. kansasii. The M. avium MICs ±50% daily omadacycline supplementation were 1mg/L and 0.5mg/L on day 7, whereas the M. kansasii MICs ±50% daily supplementation were >128mg/L and 32mg/L on day 7. Omadacycline degradation in solution leads to falsely high MICs when SGM doubling time exceed the drug degradation rates in solution. The challenge could be overcome by daily drug supplementation to account for the loss of potency, which is laborious, or perhaps stabilizing the drug from degradation. • Omadacycline concentration in solution declined ∼50% over 24hr. • M. avium and M. kansasii MICs ±50% daily omadacycline supplementation were 1mg/L versus 0.5mg/L and >128mg/L versus 32mg/L. • Omadacycline degradation leads to falsely high MICs when slow growing mycobacteria doubling time exceed the drug degradation rates in solution. [ABSTRACT FROM AUTHOR]

The development of resistance to azole antifungals used in the treatment of fungal infections can be a serious medical problem. Here, we investigate the molecular mechanisms associated with reduced susceptibility to fluconazole in clinical isolates of , showing evidence of the trailing growth phenomenon. The changes in membrane sterol composition were studied in the presence of subinhibitory fluconazole concentrations. Despite lanosterol and eburicol accumulating as the most prevalent sterols after fluconazole treatment, these ergosterol precursors still support growth of Candida isolates. The overexpression of ABC transporters was demonstrated by immunoblotting employing specific antibodies against Cdr1p and Cdr2p. The presence of a full-length 170 kDa protein Cdr1p was detected in two isolates, while a truncated form of Cdr1p with the molecular mass of 85 kDa was observed in isolate 966/3(2). Notably, Cdr2p was detected in this isolate, and the expression of this transporter was modulated by subinhibitory concentrations of fluconazole. These results suggest that C. dubliniensis can display the trailing growth phenomenon, and such isolates express similar molecular mechanisms like that of fluconazole-resistant isolates and can therefore be associated with recurrent infections. [ABSTRACT FROM AUTHOR]

A ‘trailing’ effect has been commonly observed when azole antifungals are tested against Candida spp. Previous experience with fluconazole indicates that 24-h minimum inhibitory concentration (MIC) values are more compatible endpoints when compared with clinical outcomes. We evaluated the trailing effect of Candida isolates tested with itraconazole in a guinea pig model of systemic candidiasis. Survival and organ burden were only significantly affected by using a higher dose of itraconazole, irrespective of the MIC differences at 24 and 48 h. A fluconazole-resistant strain with susceptible dose-dependent MICs to itraconazole was successfully treated with high-dose itraconazole. Our data suggests that survival and microbiological response depend more on drug dosing than on the trailing phenotype of the isolates. [ABSTRACT FROM AUTHOR]

Objective Paradoxical growth (PG) and trailing effect (TE) are frequently observed during antifungal susceptibility testing (AFST). These two phenomena interfere with the determination of the minimal inhibitory concentration (MIC). The aim of this study was to assess the clinical impact of TE and PG. Methods We analysed the frequency of TE and PG of 690 Candida isolates collected from a population-based study performed in Spain (CANDIPOP) and correlated the results with clinical outcome of the patients. Results Around 70% (484/690) of the isolates exhibited TE to azoles. Candida tropicalis showed the highest presence of TE (39/53 isolates exhibited residual growth >25% of control). No TE was seen in most of the isolates from the psilosis complex. PG was mainly associated with echinocandins. In patients treated with fluconazole within the first 48 hours after blood sampling ( n = 221), the presence of TE to azoles tended to be associated with lower 30-day mortality (odds ratio (OR) 0.55, 95% confidence interval (CI) 0.25–1.00) but not with clinical failure (OR 0.85, 95% CI 0.45–1.54). In the subgroup of 117 patients treated with echinocandins, the presence of PG was not associated with patient's response to antifungal treatment (OR for 30-day mortality 1.63, 95% CI 0.76–4.03; OR for clinical failure 1.17, 95% CI 0.53–2.70). Conclusions TE or PG are widely expressed among Candida spp., although they do not seem to influence clinical outcome. [ABSTRACT FROM AUTHOR]

Background: Emergence of resistance to respective antifungal drugs is a primary concern for the treatment of candidiasis. Hence, determining antifungal susceptibility of the isolated yeasts is of special importance for effective therapy. For this purpose, the clinical laboratory standard institute (CLSI) has introduced a broth microdilution method to determine minimum inhibitory concentration (MIC). However, the so-called "Trailing effect" phenomenon might sometimes pose ambiguity in the interpretation of the results., Objectives: The present study aimed to determine the in vitro susceptibility of clinical isolates of Candida against azoles and the frequency of the Trailing effect., Materials and Methods: A total of 193 Candida isolates were prospectively collected and identified through the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Using a broth microdilution test, according to the guidelines of CLSI M27-A3, antifungal susceptibilities of the isolated yeasts against Fluconazole (FLU), Itraconazole (ITR), Ketoconazole (KET) and Voriconazole (VOR) were assessed. Moreover, trailing growth was determined when a susceptible MIC was incubated for 24 hours, and turned into a resistant one after 48 hours of incubation., Results: Among the tested antifungal drugs in this study, the highest rate of resistance was observed against ITR (28.5%) followed by VOR (26.4%), FLU (20.8%) and KET (1.5%). The trailing effect was induced in 27 isolates (14.0%) by VOR, in 26 isolates (13.5%) by ITR, in 24 isolates (12.4%) by FLU, and in 19 isolates (9.8%) by KET., Conclusions: The monitoring of antifungal susceptibilities of Candida species isolated from clinical sources is highly recommended for the efficient management of patients. Moreover, the trailing effect should be taken into consideration once the interpretation of the results is intended.