Surveillance of Antimicrobial Resistance and Multidrug Resistance Prevalence of Clinical Isolates in a Regional Hospital in Northern Greece.

Antimicrobial resistance is a global health threat resulting in increased morbidity and mortality. This retrospective study aimed to estimate antimicrobial susceptibility and multidrug resistance prevalence of clinical isolates in a regional hospital in Northern Greece during the last 6 years by analyzing the annual reports of the Laboratory of Microbiology. A total of 12,274 strains of certain bacteria were isolated from both hospitalized and ambulatory patients from biological products, mainly urine (range 63-78% during the study period). E. coli was the most frequent pathogen found (37.4%). A significant increase in the number of the main pathogens causing hospital-acquired infections (Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterococcus faecium) was found in the time period of 2021-2023 compared to 2018-2020 (p < 0.0001). In total, 1767 multidrug-resistant bacterial strains were isolated, most of them belonging to Acinetobacter baumannii (36.4%) and Klebsiella pneumoniae (39.6%), and were located in the intensive care unit (ICU) (59.8%). Extensively drug resistance (XDR) and pan drug resistance (PDR) were significantly higher in 2021-2023 than in 2018-2020 (XDR: 641/1087 in 2021-2023 vs. 374/680 in 2018-2020 and PDR: 134/1087 in 2021-2023 vs. 25/680 in 2018-2020, p < 0.0001), resulting in an urgent need to establish certain strategies in order to eliminate this threatening condition.

Age, Sex and BMI Relations with Anti-SARS-CoV-2-Spike IgG Antibodies after BNT162b2 COVID-19 Vaccine in Health Care Workers in Northern Greece.

The aim of this work was to study age, sex, and BMI (Body Mass Index)-related differences in the development of anti-SARS-CoV-2-Spike IgG antibodies, after vaccination with the BNT162b2 COVID-19 vaccine, in health care workers of a General Hospital in a city in Northern Greece. Blood sampling was drawn two to four weeks following the second dose of the vaccine, and six months after the first blood sample collection. Measurement of serum IgG antibodies against the spike domain of SARS-CoV-2 was performed using the SARS-CoV-2 IgG II Quant assay. All participants had sufficient serum IgG titers in the first measurement. Women developed higher IgG titers than men. The IgG titers were inversely related to age in both sexes; there was also a small, insignificant tendency to be inversely related to BMI. Six months after the first measurement, the IgG titers decreased dramatically to values less than 5% of the initial. This decrease was observed in both men and women and was inversely related to age. Multivariate regression analysis showed that age and sex explained with statistical significance 9% of the variance in SARS-CoV-2 IgG titers in our study population; the role of BMI was limited and insignificant.

Clinical pharmacology of antifungal agents to overcome drug resistance in pediatric patients.

INTRODUCTION: Antifungal resistance is an emerging problem that increases morbidity and mortality in immunosuppressed pediatric patients, who suffer from invasive fungal diseases. Optimal pharmacological management is critical for the successful treatment of invasive fungal infections by resistant strains. AREAS COVERED: This paper reviews the mechanisms of resistance of different classes of antifungal agents and the current understanding of pediatric antifungal pharmacology for overcoming antifungal resistance in children based on laboratory and clinical studies in the English literature. The therapeutic choices against fungal pathogens with intrinsic or acquired resistance are further reviewed. EXPERT OPINION: There is a paucity of data in the pediatric population regarding the epidemiology of the resistant organisms to different antifungal agents. It is also unknown if there are more prevalent molecular mechanisms that promote antifungal resistance. Selection and dosages of the most effective antifungal agent for overcoming the antifungal resistance is crucial. However, there are limited studies guiding the optimal dosage and duration of treatment for management of emergent antifungal resistance. Further studies are warranted to elucidate the optimal pharmacology of the current antifungal agents against resistant organisms and to advance the development of new antifungal agents.

Deficiency of interleukin-1 receptor-associated kinase 4 presenting as fatal Pseudomonas aeruginosa bacteremia in two siblings.

Interleukin-1 receptor-associated kinase 4 (IRAK-4) deficiency is a primary immunodeficiency of innate immunity. This is the case of a previous healthy toddler and his sibling, who both died of fulminant sepsis due to Pseudomonas aeruginosa. Subsequent genetic analysis demonstrated IRAK-4 deficiency with compound heterozygous splice mutations. Fulminant fatal P. aeruginosa sepsis may be the first manifestation of IRAK-4 deficiency.

Systematic review and meta-analysis of in vitro synergy of polymyxins and carbapenems.

Our objective was to examine the evidence of in vitro synergy of polymyxin-carbapenem combination therapy against Gram-negative bacteria (GNB). A systematic review and meta-analysis were performed. All studies examining in vitro interactions of antibiotic combinations consisting of any carbapenem with colistin or polymyxin B against any GNB were used. A broad search was conducted with no language, date, or publication status restrictions. Synergy rates, defined as a fractional inhibitory concentration index of ≤0.5 or a >2-log reduction in CFU, were pooled separately for time-kill, checkerboard, and Etest methods in a mixed-effect meta-analysis of rates. We examined whether the synergy rate depended on the testing method, type of antibiotic, bacteria, and resistance to carbapenems. Pooled rates with 95% confidence intervals (CI) are shown. Thirty-nine published studies and 15 conference proceeding were included, reporting on 246 different tests on 1,054 bacterial isolates. In time-kill studies, combination therapy showed synergy rates of 77% (95% CI, 64 to 87%) for Acinetobacter baumannii, 44% (95% CI, 30 to 59%) for Klebsiella pneumoniae, and 50% (95% CI, 30 to 69%) for Pseudomonas aeruginosa, with low antagonism rates for all. Doripenem showed high synergy rates for all three bacteria. For A. baumannii, meropenem was more synergistic than imipenem, whereas for P. aeruginosa the opposite was true. Checkerboard and Etest studies generally reported lower synergy rates than time-kill studies. The use of combination therapy led to less resistance development in vitro. The combination of a carbapenem with a polymyxin against GNB, especially A. baumannii, is supported in vitro by high synergy rates, with low antagonism and less resistance development. These findings should be examined in clinical studies.

Synergistic interaction of the triple combination of amphotericin B, ciprofloxacin, and polymorphonuclear neutrophils against Aspergillus fumigatus.

Aspergillus is damaged by polymorphonuclear neutrophils (PMNs) by means of nonoxidative and oxidative mechanisms, which may be affected by antifungal and antibacterial agents that patients with invasive pulmonary aspergillosis often receive. The pharmacodynamic interactions among deoxycholate amphotericin B (AMB), ciprofloxacin (CIP), and human PMNs against Aspergillus fumigatus growth are unknown. We therefore studied the interactions between 0.032 to 2.0 µg/ml of AMB, 0.1 to 50 µg/ml of CIP at a fixed AMB/CIP ratio of 1:3.125, and PMNs from six donors at an effector-to-target (E:T) ratio of 400:1 against a clinical A. fumigatus isolate using an XTT metabolic assay and the Bliss independence pharmacodynamic-interaction model. CIP exhibited no antifungal activity alone or in combination with PMNs. Synergy was found between AMB and PMNs, with interaction indices (II) of 0.06 to 0.21; the highest interaction of 21% ± 3.6% was observed at 0.22 ± 0.09 µg/ml of AMB. The AMB and CIP (AMB+CIP) combination was synergistic (II = 0.39) at low AMB concentrations and antagonistic (II = 1.39) at high AMB concentrations, with a maximal synergistic interaction of 16% ± 3.7% observed at 0.16 ± 0.08 µg/ml of AMB. The triple combination AMB+CIP+PMNs was synergistic, with interaction indices of 0.05 to 0.20, and a maximal synergistic interaction of 24% ± 4% was observed at 0.20 ± 0.07 µg/ml of AMB. The increased percentage of Bliss synergy of the triple combination AMB+CIP+PMNs (24% ± 4%) was the product of those of the constituent double combinations AMB+PMNs (21% ± 3.6%) and AMB+CIP (16% ± 3.7%). Thus, the antifungal activity of AMB, at clinically relevant concentrations, was enhanced in combination with PMNs and CIP against A. fumigatus growth in a concentration-dependent manner.

Combination therapy in treatment of experimental pulmonary aspergillosis: in vitro and in vivo correlations of the concentration- and dose- dependent interactions between anidulafungin and voriconazole by Bliss independence drug interaction analysis.

We studied the antifungal activity of anidulafungin (AFG) in combination with voriconazole (VRC) against experimental invasive pulmonary aspergillosis (IPA) in persistently neutropenic rabbits and further explored the in vitro and in vivo correlations by using Bliss independence drug interaction analysis. Treatment groups consisted of those receiving AFG at 5 (AFG5 group) and 10 (AFG10 group) mg/kg of body weight/day, VRC at 10 mg/kg every 8 h (VRC group), AFG5 plus VRC (AFG5+VRC group), and AFG10 plus VRC (AFG10+VRC group) and untreated controls. Survival throughout the study was 60% for the AFG5+VRC group, 50% for the VRC group, 27% for the AFG10+VRC group, 22% for the AFG5 group, 18% for the AFG10 group, and 0% for control rabbits (P < 0.001). There was a significant reduction of organism-mediated pulmonary injury, measured by infarct scores, lung weights, residual fungal burdens, and galactomannan indexes, in AFG5+VRC-treated rabbits versus those treated with AFG5 and VRC alone (P < 0.05). In comparison, AFG10+VRC significantly lowered only infarct scores and lung weights in comparison to those of AFG10-treated animals (P < 0.05). AFG10+VRC showed no significant difference in other outcome variables. Significant Bliss synergy was found in vivo between AFG5 and VRC, with observed effects being 24 to 30% higher than expected levels if the drugs were acting independently. These synergistic interactions were also found between AFG and VRC in vitro. However, for AFG10+VRC, only independence and antagonism were observed among the outcome variables. We concluded that the combination of AFG with VRC in treatment of experimental IPA in persistently neutropenic rabbits was independent to synergistic at a dosage of 5 mg/kg/day but independent to antagonistic at 10 mg/kg/day, as assessed by Bliss independence analysis, suggesting that higher dosages of an echinocandin may be deleterious to the combination.

Comparative pharmacodynamic interaction analysis between ciprofloxacin, moxifloxacin and levofloxacin and antifungal agents against Candida albicans and Aspergillus fumigatus.

OBJECTIVES: Patients suffering from invasive mycoses often receive concomitant antifungal therapy and antibacterial agents. Ciprofloxacin, a carboxyfluoroquinolone, was previously observed to demonstrate the pharmacodynamic interactions with antifungal agents by altering their growth inhibitory activity against Candida albicans and Aspergillus fumigatus. However, little is known about the interaction between other extended-spectrum fluoroquinolones, such as levofloxacin and moxifloxacin, and antifungal agents against C. albicans and A. fumigatus. METHODS: Using a microdilution chequerboard technique, we employed isobolographic analysis adapted to incorporate a non-active agent in order to analyse the potential in vitro interaction between ciprofloxacin, levofloxacin or moxifloxacin and the following representative antifungal agents: amphotericin B, fluconazole or voriconazole and caspofungin. RESULTS: Synergistic interactions [interaction indices (Iis) 0.69-0.83, P < 0.05] were observed between amphotericin B (0.07-0.31 mg/L) and either ciprofloxacin (0.19-7.65 mg/L) or levofloxacin (0.41-32.88 mg/L) against C. albicans and A. fumigatus. Synergy (Iis 0.56-0.87, P < 0.05) also was found between voriconazole (0.09-0.14 mg/L) and ciprofloxacin (0.22-11.41 mg/L) as well as between caspofungin (8.94-22.07 mg/L) and levofloxacin (0.14-5.17 mg/L) against A. fumigatus. Some antagonistic (Iis 1.16-1.29, P < 0.05) interactions were observed between fluoroquinolones and fluconazole against C. albicans. In general, ciprofloxacin enhanced the activity of antifungal agents more than moxifloxacin and levofloxacin against both C. albicans and A. fumigatus. CONCLUSIONS: The knowledge of the pharmacodynamic interactions between fluoroquinolones and antifungal agents may guide selection and potentially improve the outcome of immunosuppressed patients with concurrent bacterial and fungal infections.

In vitro activity of CAY-1, a saponin from Capsicum frutescens, against Microsporum and Trichophyton species.

Dermatomycoses are among the world's most common diseases and their incidence has increased over recent years, particularly in immunosuppressed patients. In previous studies, the saponin CAY-1 from cayenne pepper (Capsicum frutescens), has shown antifungal activities against Candida albicans and Aspergillus spp. We therefore studied the in vitro antifungal activity of CAY-1 against non-germinating conidia and hyphae of clinical isolates of the dermatophytes Trichophyton mentagrophytes, T. rubrum, T. tonsurans and Microsporum canis. We used a microdilution method to assess the growth inhibitory activities of CAY-1 against conidia (CLSI document M38-A) and a colorimetric procedure (XTT method) to investigate the metabolic inhibitory activity of CAY-1 against hyphae. The minimal inhibitory concentrations (complete visual growth inhibition) of CAY-1 against non-germinating conidia ranged from 10-20 microg/ml for all dermatophyte isolates included in this investigation. In addition, we found >90% inhibition of hyphal metabolic activity of these same isolates with 10-20 microg/ml of CAY-1. Results indicate that CAY-1 merits further investigation as a potential agent for the treatment of dermatomycoses.

Isobolographic analysis of pharmacodynamic interactions between antifungal agents and ciprofloxacin against Candida albicans and Aspergillus fumigatus.

Patients suffering from invasive mycoses often receive concomitant antifungal therapy and antibacterial agents. Assessment of pharmacodynamic interactions between antifungal and antibacterial agents is complicated by the absence of a common antifungal end point for both agents. Ciprofloxacin has no intrinsic antifungal activity but may interact with antifungal agents, since it inhibits DNA gyrase (topoisomerase II), which is abundant in fungi. We therefore employed isobolographic analysis adapted to incorporate a nonactive agent in order to analyze the potential in vitro interaction between the fluoroquinolone ciprofloxacin and several representative antifungal agents against Candida albicans and Aspergillus fumigatus strains by using a microdilution checkerboard technique. In agreement with earlier in vitro studies, conventional fractional inhibitory concentration index analysis was unable to detect interactions between ciprofloxacin and antifungal agents. However, isobolographic analysis revealed significant pharmacodynamic interactions between antifungal agents and ciprofloxacin against C. albicans and A. fumigatus strains. Amphotericin B demonstrated concentration-dependent interactions for both species, with synergy (interaction indices, 0.14 to 0.81) observed at ciprofloxacin concentrations of <10.64 microg/ml. Synergy (interaction indices, 0.10 to 0.86) was also found for voriconazole and caspofungin against A. fumigatus. Isobolographic analysis may help to elucidate the pharmacodynamic interactions between antifungal and non-antifungal agents and to develop better management strategies against invasive candidiasis and aspergillosis.

Differential fungicidal activities of amphotericin B and voriconazole against Aspergillus species determined by microbroth methodology.

Antifungal agents may differ in their fungicidal activities against Aspergillus spp. In order to compare the fungicidal activities of voriconazole and amphotericin B against 40 isolates of Aspergillus fumigatus, A. flavus, and A. terreus, we developed a new microbroth colorimetric method for assessing fungicidal activities and determining minimal fungicidal concentrations (MFCs). This methodology follows the antifungal susceptibility testing reference method M-38A for MIC determination. After drug removal and addition of fresh medium, growth of viable conidia adhering to the bottoms of the microtitration wells was assessed by a colorimetric assay of metabolic activity after 24 h of incubation. The new method was faster (six times), reproducible (92 to 97%), and in agreement with culture-based MFCs (91 to 100%). Differential fungicidal activities of voriconazole and amphotericin B were found among the three Aspergillus species, with A. fumigatus and A. flavus having the lowest (1 and 2 mg/liter, respectively) and A. terreus the highest (>16 mg/liter) median amphotericin B MFCs; A. flavus had a lower median voriconazole MFC (4 mg/liter) than the other species (>8 mg/liter; P < 0.05). Amphotericin B was fungicidal (MFC/MIC 4) against 94% of A. fumigatus and 84% of A. terreus isolates. The new methodology revealed a concentration-dependent sigmoid pattern of fungicidal effects, indicating that fungicidal activity is not an all-or-nothing phenomenon and that some degree of fungicidal action can be found even for agents considered fungistatic based on the MFC/MIC ratio.

Concentration-dependent synergy and antagonism within a triple antifungal drug combination against Aspergillus species: analysis by a new response surface model.

Triple antifungal combinations are used against refractory invasive aspergillosis without an adequate understanding of their pharmacodynamic interactions. We initially studied the in vitro triple combination of voriconazole, amphotericin B, and caspofungin against Aspergillus fumigatus, A. flavus, and A. terreus by a spectrophotometric microdilution broth method after 48 h of incubation. We then analyzed these results with a recently described nonlinear mixture response surface E(max)-based model modified to assess pharmacodynamic interactions at various growth levels. The new model allows flexibility in all four parameters of the E(max) model and is able to describe complex pharmacodynamic interactions. Concentration-dependent pharmacodynamic interactions were found within the triple antifungal combination. At the 50% growth level, synergy (median interaction indices of 0.43 to 0.82) was observed at low concentrations of voriconazole (<0.03 mg/liter) and amphotericin B (

Host-dependent patterns of tissue injury in invasive pulmonary aspergillosis.

Invasive pulmonary aspergillosis (IPA) is an important cause of morbidity and mortality in neutropenic, nonneutropenic, and other immunocompromised patients. We therefore compared the patterns of infection and inflammation among 3 cohorts of immunocompromised patients with profound neutropenia, nonneutropenic immunosuppression, and hematopoietic stem cell transplantation. Lesions of IPA in neutropenic patients and hematopoietic stem cell transplant (HSCT) recipients were similar and consisted predominantly of angioinvasion and intraalveolar hemorrhage. The frequency of these histologic findings in neutropenic patients and HSCT recipients differed significantly from those of nonneutropenic patients (P < .05). It is noteworthy that even if HSCT recipients have normal peripheral blood neutrophil counts, there may be no influx into sites of infection. In the nonneutropenic cohort, lesions of IPA consisted mainly of neutrophilic and monocytic infiltrates and inflammatory necrosis. Thus, the status of innate host defenses contributes significantly to the histologic patterns observed in IPA.

Antifungal interactions within the triple combination of amphotericin B, caspofungin and voriconazole against Aspergillus species.

OBJECTIVES: The in vitro effects of caspofungin combined with voriconazole and amphotericin B were tested in triplicate experiments against nine clinical isolates of Aspergillus fumigatus, Aspergillus flavus and Aspergillus terreus. METHODS: The isolates were tested against a range of concentrations of voriconazole (0.015-1.0 mg/L), caspofungin (0.125-256 mg/L) and five concentrations of amphotericin B (0.1-0.5 mg/L) with a microdilution chequerboard method based on the CLSI M38-A reference method and the results were analysed with the fractional inhibitory concentration (FIC) index. The effect of individual drugs on the FIC index of each of the double combinations was also evaluated. RESULTS: The triple combination of voriconazole, caspofungin and amphotericin B against all Aspergillus spp. was synergistic (FIC index 0.49-0.57) at low median concentrations of amphotericin B (0.10-0.22 mg/L) and voriconazole (0.07-0.15 mg/L) over a wide range of caspofungin concentrations (4.32-17.28 mg/L). Antagonistic interactions (FIC index 1.65-2.15) were found at higher median concentrations of amphotericin B (0.3-0.5 mg/L) and voriconazole (0.23-0.68 mg/L) over a similarly wide range of caspofungin concentrations (1.47-32 mg/L). CONCLUSIONS: These concentration-dependent interactions may have important clinical implications, which require further evaluation in animal models of invasive aspergillosis.

Triazole-polyene antagonism in experimental invasive pulmonary aspergillosis: in vitro and in vivo correlation.

Combination antifungal therapy is increasingly used in the treatment of invasive aspergillosis. Whether the interaction between amphotericin B and triazoles is antagonistic against invasive aspergillosis is a controversial issue that is not likely to be resolved through a randomized clinical trial. Here, we found both in vitro and in vivo antagonism between liposomal amphotericin B and ravuconazole in simultaneous treatment of experimental invasive pulmonary aspergillosis in persistently neutropenic rabbits. Bliss independence-based drug-interaction modeling showed significant antagonism in vitro and in vivo, with the observed drug effects being 20%-69% lower than would be expected if the drugs were acting independently. These in vitro and in vivo findings of antagonism were consistent with the findings from Loewe additivity-based drug-interaction modeling. No pharmacokinetic interaction was found. The combination of a triazole and polyene may be antagonistic in the treatment of invasive pulmonary aspergillosis.

Deoxycholate amphotericin B and amphotericin B lipid complex exert additive antifungal activity in combination with pulmonary alveolar macrophages against Fusarium solani.

Fusarium spp. have emerged as important causes of invasive fungal infections in immunocompromised patients. Rabbit pulmonary alveolar macrophages (PAMs) exhibited fungicidal activity against conidia of Fusarium solani and achieved a time-dependent increase in killing. Neither deoxycholate amphotericin B (DAMB) nor amphotericin B lipid complex (ABLC) exerted a suppressive effect on PAMs by decreasing their conidiocidal activity against F. solani. On the contrary, at a concentration of 0.125 microg ml(-1), ABLC and, to a lesser degree, DAMB additively augmented the fungicidal activity of pulmonary alveolar macrophages against conidia of Fusarium solani.

Micafungin: pharmacology, experimental therapeutics and clinical applications.

Invasive fungal infections are important causes of morbidity and mortality in hospitalised patients. Current therapy with amphotericin B and antifungal triazoles has overlapping targets and is limited by toxicity and resistance. Echinocandins are a new class of antifungal drugs, which inhibit the synthesis of 1,3-beta-D-glucan. This homopolysaccharide is an important component of the cell wall of many pathogenic fungi, providing osmotic stability and functioning in cell growth and cell division. Micafungin, which is a member of the echinocandin class, exhibits in vitro fungicidal or fungistatic activity against a variety of fungal pathogens which include Candida and Aspergillus species but not Cryptococcus, Fusarium or Zygomycetes. Micafungin demonstrates linear pharmacokinetics, which are not altered by drugs metabolised through the P450 enzyme system. The preclinical and clinical data strongly support the development of micafungin for treatment of proven or suspected mucosal and invasive Candida infections in immunocompetent and immunocompromised patients. This paper reviews the preclinical and clinical pharmacology of micafungin and its potential role for treatment of fungal invasive infections in patients.

Human phagocytic cell responses to Scedosporium apiospermum (Pseudallescheria boydii): variable susceptibility to oxidative injury.

Scedosporium apiospermum (Pseudallescheria boydii) is an emerging opportunistic filamentous fungus that causes serious infections in both immunocompetent and immunocompromised patients. To gain insight into the immunopathogenesis of infections due to S. apiospermum, the antifungal activities of human polymorphonuclear leukocytes (PMNs), mononuclear leukocytes (MNCs), and monocyte-derived macrophages (MDMs) against two clinical isolates of S. apiospermum were evaluated. Isolate SA54A was amphotericin B resistant and was the cause of a fatal disseminated infection. Isolate SA1216 (cultured from a successfully treated localized subcutaneous infection) was susceptible to amphotericin B. MDMs exhibited similar phagocytic activities against conidia of both isolates. However, PMNs and MNCs responded differently to the hyphae of these two isolates. Serum opsonization of hyphae resulted in a higher level of superoxide anion (O(2)(-)) release by PMNs in response to SA54A (amphotericin B resistant) than that seen in response to SA1216 (amphotericin B susceptible; P < 0.001). Despite this increased O(2)(-) production, PMNs and MNCs induced less hyphal damage to SA54A than to SA1216 (P < 0.001). To investigate the potential mechanisms responsible for these differences, hyphal damage was evaluated in the presence of antifungal oxidative metabolites as well as in the presence of a series of inhibitors and scavengers of antifungal PMN function. Mannose, catalase, superoxide dismutase, dimethyl sulfoxide, and heparin had no effect on PMN-induced hyphal damage to either of the two isolates. However, azide, which inhibits PMN myeloperoxidase activity, significantly reduced hyphal damage to SA1216 (P < 0.01) but not to SA54A. Hyphae of SA1216 were slightly more susceptible to oxidative pathway products, particularly HOCl, than those of SA54A. Thus, S. apiospermum is susceptible to antifungal phagocytic function to various degrees. The selective inhibitory pattern of azide with respect to hyphal damage and the parallel susceptibility to HOCl suggests an important difference in susceptibilities to myeloperoxidase products that may be related to the various levels of pathogenicity and amphotericin B resistance of S. apiospermum.