Pharmacokinetics and pharmacodynamics of bacteriophage therapy: a review with a focus on multidrug-resistant Gram-negative bacterial infections.

SUMMARYDespite the early recognition of their therapeutic potential and the current escalation of multidrug-resistant (MDR) pathogens, the adoption of bacteriophages into mainstream clinical practice is hindered by unfamiliarity with their basic pharmacokinetic (PK) and pharmacodynamic (PD) properties, among others. Given the self-replicative nature of bacteriophages in the presence of host bacteria, the adsorption rate, and the clearance by the host's immunity, their PK/PD characteristics cannot be estimated by conventional approaches, and thus, the introduction of new considerations is required. Furthermore, the multitude of different bacteriophage types, preparations, and treatment schedules impedes drawing general conclusions on their in vivo PK/PD features. Additionally, the drawback of acquired bacteriophage resistance of MDR pathogens with clinical and environmental implications should be taken into consideration. Here, we provide an overview of the current state of the field of PK and PD of bacteriophage therapy with a focus on its application against MDR Gram-negative infections, highlighting the potential knowledge gaps and the challenges in translation from the bench to the bedside. After reviewing the in vitro PKs and PDs of bacteriophages against the four major MDR Gram-negative pathogens, Klebsiella pneumoniae, Acinetobacter baumannii complex, Pseudomonas aeruginosa, and Escherichia coli, specific data on in vivo PKs (tissue distribution, route of administration, and basic PK parameters in animals and humans) and PDs (survival and reduction of bacterial burden in relation to the route of administration, timing of therapy, dosing regimens, and resistance) are summarized. Currently available data merit close scrutiny, and optimization of bacteriophage therapy in the context of a better understanding of the underlying PK/PD principles is urgent to improve its therapeutic effect and to minimize the occurrence of bacteriophage resistance.

Increase in candidemia cases and emergence of fluconazole-resistant Candida parapsilosis and C. auris isolates in a tertiary care academic hospital during the COVID-19 pandemic, Greece, 2020 to 2023.

BackgroundThe COVID-19 pandemic and the emergence of Candida auris have changed the epidemiological landscape of candidaemia worldwide.AimWe compared the epidemiological trends of candidaemia in a Greek tertiary academic hospital before (2009-2018) and during the early COVID-19 (2020-2021) and late COVID-19/early post-pandemic (2022-2023) era.MethodsIncidence rates, species distribution, antifungal susceptibility profile and antifungal consumption were recorded, and one-way ANOVA or Fisher's exact test performed. Species were identified by MALDI-ToF MS, and in vitro susceptibility determined with CLSI M27-Ed4 for C. auris and the EUCAST-E.DEF 7.3.2 for other Candida spp.ResultsIn total, 370 candidaemia episodes were recorded during the COVID-19 pandemic. Infection incidence (2.0 episodes/10,000 hospital bed days before, 3.9 during the early and 5.1 during the late COVID-19 era, p < 0.0001), C. auris (0%, 9% and 33%, p < 0.0001) and fluconazole-resistant C. parapsilosis species complex (SC) (20%, 24% and 33%, p = 0.06) infections increased over time, with the latter not associated with increase in fluconazole/voriconazole consumption. A significant increase over time was observed in fluconazole-resistant isolates regardless of species (8%, 17% and 41%, p < 0.0001). Resistance to amphotericin B or echinocandins was not recorded, with the exception of a single pan-echinocandin-resistant C. auris strain.ConclusionCandidaemia incidence nearly tripled during the COVID-19 era, with C. auris among the major causative agents and increasing fluconazole resistance in C. parapsilosis SC. Almost half of Candida isolates were fluconazole-resistant, underscoring the need for increased awareness and strict implementation of infection control measures.

A single-layer spot assay for easy, fast, and high-throughput quantitation of phages against multidrug-resistant Gram-negative pathogens.

Double-layer agar (DLA) overlay plaque assay is the gold standard for phage enumeration. However, it is cumbersome and time-consuming. Given the great interest in phage therapy, we explored alternative assays for phage quantitation. A total of 16 different phages belonging to Myoviridae, Siphoviridae, and Podoviridae families were quantitated with five K. pneumoniae, eight P. aeruginosa, and three A. baumannii host isolates. Phages were quantitated with the standard DLA assay (10 mL of LB soft agar 0.7% on LB hard agar 1.5%) and the new single-layer agar (SLA) assay (10 mL of LB soft agar 0.7%) with phages spread (spread) into or spotted (spot) onto soft agar. Phage concentrations with each assay were correlated with the standard assay, and the relative and absolute differences between each assay and the standard double-layer agar spread were calculated. Phage concentrations 1 × 104-8.3 x1012 PFU/mL with the standard DLA assay were quantitated with SLA-spread, SLA-spot, and DLA-spot assays, and the median (range) relative and absolute differences were <10% and <0.98 log10PFU/mL, respectively, for all phage/bacterial species (ANOVA P = 0.1-0.43), and they were highly correlated (r > 0.77, P < 0.01). Moreover, plaques could be quantified at 37°C after 4-h incubation for K. pneumoniae phages and 6-h incubation for P. aeruginosa and A. baumannii phages, and estimated concentrations remained the same over 24 hours. Compared to DLA assay, the SLA-spot assay required less media, it was 10 times faster, and generated same-day results. The SLA-spot assay was cheaper, faster, easier to perform, and generated similar phage concentrations as the standard DLA-spread assay.

Comparative pharmacodynamics and dose optimization of liposomal amphotericin B against Candida species in an in vitro pharmacokinetic/pharmacodynamic model.

As comparative pharmacokinetic/pharmacodynamic (PK/PD) studies of liposomal amphotericin B (L-AMB) against Candida spp. are lacking, we explored L-AMB pharmacodynamics against different Candida species in an in vitro PK/PD dilution model. Eight Candida glabrata, Candida parapsilosis, and Candida krusei isolates (EUCAST/CLSI AMB MIC 0.125-1 mg/L) were studied in the in vitro PK/PD model simulating L-AMB Cmax = 0.25-64 mg/L and t1/2 = 9 h. The model was validated with one susceptible and one resistant Candida albicans isolate. The Cmax/MIC-log10CFU/mL reduction from the initial inoculum was analyzed with the Emax model, and Monte Carlo analysis was performed for the standard (3 mg/kg with Cmax = 21.87 ± 12.47 mg/L) and higher (5 mg/kg with Cmax = 83 ± 35.2 mg/L) L-AMB dose. A ≥1.5 log10CFU/mL reduction was found at L-AMB Cmax = 8 mg/L against C. albicans, C. parapsilosis, and C. krusei isolates (MIC 0.25-0.5 mg/L) whereas L-AMB Cmax ≥ 32 mg/L was required for C. glabrata isolates. The in vitro PK/PD relationship followed a sigmoidal pattern (R2 ≥ 0.85) with a mean Cmax/MIC required for stasis of 2.1 for C. albicans (close to the in vivo stasis), 24/17 (EUCAST/CLSI) for C. glabrata, 8 for C. parapsilosis, and 10 for C. krusei. The probability of target attainment was ≥99% for C. albicans wild-type (WT) isolates with 3 mg/kg and for wild-type isolates of the other species with 5 mg/kg. L-AMB was four- to eightfold less active against the included non-C. albicans species than C. albicans. A standard 3-mg/kg dose is pharmacodynamically sufficient for C. albicans whereas our data suggest that 5 mg/kg may be recommendable for the included non-C. albicans species.

Candida auris in Greek healthcare facilities: Active surveillance results on first cases and outbreaks from eleven hospitals within Attica region.

BACKGROUND: Candida auris was sporadically detected in Greece until 2019. Thereupon, there has been an increase in isolations among inpatients of healthcare facilities. AIM: We aim to report active surveillance data on MALDI-TOF confirmed Candida auris cases and outbreaks, from November 2019 to September 2021. METHODS: A retrospective study on hospital-based Candida auris data, over a 23-month period was conducted, involving 11 hospitals within Attica region. Antifungal susceptibility testing and genotyping were conducted. Case mortality and fatality rates were calculated and p-values less than 0.05 were considered statistically significant. Infection control measures were enforced and enhanced. RESULTS: Twenty cases with invasive infection and 25 colonized were identified (median age: 72 years), all admitted to hospitals for reasons other than fungal infections. Median hospitalisation time until diagnosis was 26 days. Common risk factors among cases were the presence of indwelling devices (91.1 %), concurrent bacterial infections during hospitalisation (60.0 %), multiple antimicrobial drug treatment courses prior to hospitalisation (57.8 %), and admission in the ICU (44.4 %). Overall mortality rate was 53 %, after a median of 41.5 hospitalisation days. Resistance to fluconazole and amphotericin B was identified in 100 % and 3 % of tested clinical isolates, respectively. All isolates belonged to South Asian clade I. Outbreaks were identified in six hospitals, while remaining hospitals detected sporadic C. auris cases. CONCLUSION: Candida auris has proven its ability to rapidly spread and persist among inpatients and environment of healthcare facilities. Surveillance focused on the presence of risk factors and local epidemiology, and implementation of strict infection control measures remain the most useful interventions.

Evaluation of the Vitek 2 system for antifungal susceptibility testing of Candida auris using a representative international panel of clinical isolates: overestimation of amphotericin B resistance and underestimation of fluconazole resistance.

Although the Vitek 2 system is broadly used for antifungal susceptibility testing of Candida spp., its performance against Candida auris has been assessed using limited number of isolates recovered from restricted geographic areas. We therefore compared Vitek 2 system with the reference Clinical and Laboratory Standards Institute (CLSI) broth microdilution method using an international collection of 100 C. auris isolates belonging to different clades. The agreement ±1 twofold dilution between the two methods and the categorical agreement (CA) based on the Centers for Disease Control and Prevention's (CDC's) tentative resistance breakpoints and Vitek 2-specific wild-type upper limit values (WT-ULVs) were determined. The CLSI-Vitek 2 agreement was poor for 5-flucytosine (0%), fluconazole (16%), and amphotericin B (29%), and moderate for voriconazole (61%), micafungin (67%), and caspofungin (81%). Significant interpretation errors were recorded using the CDC breakpoints for amphotericin B (31% CA, 69% major errors; MaEs) and fluconazole (69% CA, 31% very major errors; VmEs), but not for echinocandins (99% CA, 1% MaEs for both micafungin and caspofungin) for which the Vitek 2 allowed correct categorization of echinocandin-resistant FKS1 mutant isolates. Discrepancies were reduced when the Vitek 2 WT-ULV of 16 mg/L for amphotericin B (98% CA, 2% MaEs) and of 4 mg/L for fluconazole (96% CA, 1% MaEs, 3% VmEs) were used. In conclusion, the Vitek 2 system performed well for echinocandin susceptibility testing of C .auris. Resistance to fluconazole was underestimated whereas resistance to amphotericin B was overestimated using the CDC breakpoints of ≥32 and ≥2 mg/L, respectively. Vitek 2 minimun inhibitory concentrations (MICs) >4 mg/L indicated resistance to fluconazole and Vitek 2 MICs ≤16 mg/L indicated non-resistance to amphotericin B.

Multidrug-resistant organism bloodstream infection and hospital acquisition among inpatients in three tertiary Greek hospitals during the COVID-19 era.

From 2019 (pre-COVID-19) to 2022 (COVID-19 years), three tertiary Greek hospitals monitored MDRO bloodstream infection (BSI) and hospital acquisition relying on laboratory data. Surveillance covered carbapenem-resistant Enterobacterales (CRE), Acinetobacter baumannii (CRAB), Pseudomonas aeruginosa (CRPA), vancomycin-resistant enterococci (VRE), and methicillin-resistant Staphylococcus aureus (MRSA), in intensive care units (ICUs) and non-ICUs. Non-ICUs experienced significant increases in CRE, CRAB and VRE during the pandemic. In ICUs, CRE increased in 2021, CRAB in 2020 and 2021, and VRE in 2021 and 2022. KPC predominated among CRE. MDRO BSI and hospital acquisition incidence rates increased, driven by CRE and CRAB.

A multicentre evaluation of the NG-test DetecTool OXA-23 for the rapid detection of OXA-23 carbapenemase directly from blood cultures.

Objectives: A multicentre study evaluating NG-Test DetecTool OXA-23 for the detection of OXA-23 carbapenemase directly from positive blood cultures (PBCs). Methods: The NG-Test DetecTool OXA-23 is an immunoassay that integrates a sample preparation device. We evaluated NG-Test DetecTool OXA-23 on 189 spiked and 126 clinical PBCs. The clinical samples' standard-of-care procedure consisted of bacterial identification from the first day of positivity by MALDI-TOF MS, conventional culture and antimicrobial susceptibility testing. The immunoassay results were verified molecularly. The strains used for the spiked samples consisted of well-characterized Acinetobacter baumannii and Proteus mirabilis strains. Results: The NG-Test DetecTool OXA-23 was evaluated on 315 PBCs and revealed sensitivity of 100% (95% CI: 98.21%-100.00%) and specificity of 100% (95% CI: 96.73%-100.00%). It provided 204 true-positive results for OXA-23 in 196 bottles with carbapenem-resistant A. baumannii (CRAB) and 8 bottles with carbapenem-resistant P. mirabilis and also provided 111 true-negative results. There were no false-positive and no false-negative results. Among the 315 PBCs studied, 83 clinical blood cultures collected in the ICU of a Greek university hospital, which were tested prospectively, all yielded CRAB, and OXA-23 was correctly detected in all samples from the first day of positivity using the NG-Test DetecTool OXA-23. Conclusions: The NG-Test DetecTool OXA-23 has exhibited excellent sensitivity and specificity for OXA-23 detection in PBCs and can provide valuable information for appropriate selection of antibiotic therapy and early implementation of infection control measures.

Antibiotic resistance in Campylobacter jejuni and Campylobacter coli: significant contribution of an RND type efflux pump in erythromycin resistance.

In this study, we aimed to determine the antibiotic resistance status of Campylobacter spp. isolated from human infections in our region, including the role of mechanisms involved in erythromycin resistance. Standard methods were used for the isolation, identification and antibiotic susceptibility testing of Campylobacter spp. isolates. Erythromycin-resistant mutants were selected from erythromycin-susceptible clinical isolates, and the erythromycin resistance mechanisms were investigated phenotypically by determining the erythromycin MICs of isolates in the presence and absence of the resistance nodulation cell division (RND) type efflux pump inhibitor, phenylalanine-arginine ß-naphthylamide dihydrochloride (PAßN), and genotypically by determining ribosomal and cmeABC alterations using PCR and DNA sequence analysis. Campylobacter spp., including 184 C. jejuni and 20 C. coli in a two-year period, were the most frequently isolated gastrointestinal bacterial pathogens in our region. However, in both C. jejuni and C. coli, resistance to tetracycline and ciprofloxacin were found to be high, erythromycin resistance was especially high (20%) in C. coli. With a ribosomal alteration, A2075G, which was found to be associated with high-level erythromycin resistance in clinical isolates, PAßN significantly reduced the erythromycin MICs in both clinical isolates and mutants. An important finding of this study, while considering cmeABC operon, is the explanation of why erythromycin resistance is more common among C. coli than C. jejuni, bearing in mind the specific deletions and alterations in the intergenic region of the operon in all erythromycin-resistant C. coli isolates. Ultimately, these findings revealed the significant role of RND-type efflux activity in increased erythromycin MICs of the isolates.

A new PK/PD target for assessing efficacy of micafungin against Candida parapsilosis.

BACKGROUND: Pharmacokinetic/pharmacodynamic (PK/PD) targets of echinocandins failed to support current clinical breakpoints of Candida parapsilosis as the PTA is low for susceptible isolates despite the good clinical efficacy of echinocandins against these infections. We therefore investigated the effect of micafungin against C. parapsilosis using an in vitro PK/PD in the presence of 10% human serum. METHODS: Three susceptible (MIC = 0.5-2 mg/L) and one resistant (MIC > 8 mg/L) C. parapsilosis sensu stricto isolates were tested at two different inocula (104 and 103 cfu/mL) simulating micafungin human exposures in RPMI and in RPMI + 10% pooled human serum. The exposure-effect relationship tAUC0-24/MIC was described and different PK/PD targets were determined in order to calculate the PTA for the standard 100 mg IV q24h dose. RESULTS: A maximal effect was found at fCmax ≥ 4 mg/L in RPMI and tCmax ≥ 64 mg/L (fCmax = 0.08 mg/L) in the presence of serum for which in vitro PK/PD targets were 50 times lower. Stasis in the presence of serum was found at 272-240 tAUC0-24/MIC, close to the clinical PK/PD target (285 tAUC/MIC), validating the in vitro model. However, the PTA was low for susceptible isolates with EUCAST/CLSI MICs ≤ 2 mg/L. Among the different PK/PD targets investigated, the PK/PD target 28 tAUC/MIC associated with 10% of maximal effect with the low inoculum resulted in PTAs ≥ 95% for susceptible isolates with EUCAST/CLSI MICs ≤ 2 mg/L. CONCLUSIONS: A new PK/PD target was found for micafungin and C. parapsilosis that supports the current clinical breakpoint. This target could be used for assessing echinocandin efficacy against C. parapsilosis.

Sustained cell-mediated but not humoral responses in rituximab-treated rheumatic patients after vaccination against SARS-CoV-2.

OBJECTIVES: B-cell depleting monoclonal antibodies are associated with increased COVID-19 severity and impaired immune response to vaccination. We aimed to assess the humoral and cell mediated (CMI) immune response after SARS-CoV-2 vaccination in rituximab (RTX)-treated rheumatic patients. METHODS: Serum and whole blood samples were collected from RTX-treated rheumatic patients 3-6 months after last vaccination against SARS-CoV-2. Serum was tested by ELISA for quantitative detection of anti-spike SARS-CoV-2 IgG. Cell-mediated variant-specific SARS-CoV-2 immunity (CMI) was assessed by interferon-γ release assay Covi-FERON FIA. Patients were interviewed for breakthrough COVID-19 infection (BTI) 3 months post sampling. RESULTS: Sixty patients were studied after a median (IQR) of 179 (117-221.5) days from last vaccine to sampling. Forty (66.7%) patients had positive Covi-FERON and 23 (38.3%) had detectable anti-spike IgG. Covi-FERON positive patients had lower median RTX cumulative dose [6 (4-10.75) vs 11 (6.75-14.75) grams, (P = 0.019)]. Patients with positive anti-spike IgG had received fewer RTX cycles [2 (2-4) vs 6 (4-8), P = 0.002] and cumulative dose [4 (3-7) vs 10 (6.25-13) grams, P = 0.002] and had shorter time from last vaccination to sampling [140 (76-199) vs 192 (128-230) days, P = 0.047]. Thirty-seven percent were positive only for Covi-FERON and 7% only for anti-spike IgG. Twenty (33.3%) BTI occurred post sampling, exclusively during Omicron variant predominance. The proportion of patients with CMI response against Delta variant was lower in patients who experienced BTI (25% vs 55%, P = 0.03). CONCLUSIONS: Four out of ten RTX-treated vaccinated patients show lasting cell-mediated immune response despite undetectable anti-spike antibodies. Cumulative RTX dose affects both humoral and cell-mediated responses to SARS-CoV-2 vaccines. Cell-mediated immune responses call for attention as a vaccine efficacy marker against SARS-CoV-2.

Pseudomonasaeruginosa antimicrobial susceptibility profiles, resistance mechanisms and international clonal lineages: update from ESGARS-ESCMID/ISARPAE Group.

SCOPE: Pseudomonas aeruginosa, a ubiquitous opportunistic pathogen considered one of the paradigms of antimicrobial resistance, is among the main causes of hospital-acquired and chronic infections associated with significant morbidity and mortality. This growing threat results from the extraordinary capacity of P. aeruginosa to develop antimicrobial resistance through chromosomal mutations, the increasing prevalence of transferable resistance determinants (such as the carbapenemases and the extended-spectrum ß-lactamases), and the global expansion of epidemic lineages. The general objective of this initiative is to provide a comprehensive update of P. aeruginosa resistance mechanisms, especially for the extensively drug-resistant (XDR)/difficult-to-treat resistance (DTR) international high-risk epidemic lineages, and how the recently approved ß-lactams and ß-lactam/ß-lactamase inhibitor combinations may affect resistance mechanisms and the definition of susceptibility profiles. METHODS: To address this challenge, the European Study Group for Antimicrobial Resistance Surveillance (ESGARS) from the European Society of Clinical Microbiology and Infectious Diseases launched the 'Improving Surveillance of Antibiotic-Resistant Pseudomonas aeruginosa in Europe (ISARPAE)' initiative in 2022, supported by the Joint programming initiative on antimicrobial resistance network call and included a panel of over 40 researchers from 18 European Countries. Thus, a ESGARS-ISARPAE position paper was designed and the final version agreed after four rounds of revision and discussion by all panel members. QUESTIONS ADDRESSED IN THE POSITION PAPER: To provide an update on (a) the emerging resistance mechanisms to classical and novel anti-pseudomonal agents, with a particular focus on ß-lactams, (b) the susceptibility profiles associated with the most relevant ß-lactam resistance mechanisms, (c) the impact of the novel agents and resistance mechanisms on the definitions of resistance profiles, and (d) the globally expanding XDR/DTR high-risk lineages and their association with transferable resistance mechanisms. IMPLICATION: The evidence presented herein can be used for coordinated epidemiological surveillance and decision making at the European and global level.

Defining Optimal Doses of Liposomal Amphotericin B Against Candida auris: Data From an In Vitro Pharmacokinetic/Pharmacodynamic Model.

BACKGROUND: Candida auris isolates exhibit elevated amphotericin B (AMB) minimum inhibitory concentrations (MICs). As liposomal AMB (L-AMB) can be safely administered at high doses, we explored L-AMB pharmacodynamics against C. auris isolates in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) dilution model. METHODS: Four C. auris isolates with Clinical and Laboratory Standards Institute (CLSI) AMB MICs = 0.5-2 mg/L were tested in an in vitro PK/PD model simulating L-AMB pharmacokinetics. The in vitro model was validated using a Candida albicans isolate tested in animals. The peak concentration (Cmax)/MIC versus log10 colony-forming units (CFU)/mL reduction from the initial inoculum was analyzed with the sigmoidal model with variable slope (Emax model). Monte Carlo analysis was performed for the standard (3 mg/kg) and higher (5 mg/kg) L-AMB doses. RESULTS: The in vitro PK/PD relationship Cmax/MIC versus log10 CFU/mL reduction followed a sigmoidal pattern (R2 = 0.91 for C. albicans, R2 = 0.86 for C. auris). The Cmax/MIC associated with stasis was 2.1 for C. albicans and 9 for C. auris. The probability of target attainment was >95% with 3 mg/kg for wild-type C. albicans isolates with MIC ≤2 mg/L and C. auris isolates with MIC ≤1 mg/L whereas 5 mg/kg L-AMB is needed for C. auris isolates with MIC 2 mg/L. CONCLUSIONS: L-AMB was 4-fold less active against C. auris than C. albicans. Candida auris isolates with CLSI MIC 2 mg/L would require a higher L-AMB dose.

Cefiderocol and Sulbactam-Durlobactam against Carbapenem-Resistant Acinetobacter baumannii.

Infections caused by carbapenem-resistant Acinetobacter baumannii (CRAB) remain a clinical challenge due to limited treatment options. Recently, cefiderocol, a novel siderophore cephalosporin, and sulbactam-durlobactam, a bactericidal ß-lactam-ß-lactamase inhibitor combination, have been approved by the Food and Drug Administration for the treatment of A. baumannii infections. In this review, we discuss the mechanisms of action of and resistance to cefiderocol and sulbactam-durlobactam, the antimicrobial susceptibility of A. baumannii isolates to these drugs, as well as the clinical effectiveness of cefiderocol and sulbactam/durlobactam-based regimens against CRAB. Overall, cefiderocol and sulbactam-durlobactam show an excellent antimicrobial activity against CRAB. The review of clinical studies evaluating the efficacy of cefiderocol therapy against CRAB indicates it is non-inferior to colistin/other treatments for CRAB infections, with a better safety profile. Combination treatment is not associated with improved outcomes compared to monotherapy. Higher mortality rates are often associated with prior patient comorbidities and the severity of the underlying infection. Regarding sulbactam-durlobactam, current data from the pivotal clinical trial and case reports suggest this antibiotic combination could be a valuable option in critically ill patients affected by CRAB infections, in particular where no other antibiotic appears to be effective.

Optimization of the EUCAST reference broth microdilution method for echinocandin susceptibility testing of Aspergillus fumigatus.

BACKGROUND: Because of the high inoculum (105 cfu/mL) used in the EUCAST susceptibility testing of Aspergillus spp., determination of the minimal effective concentration (MEC) of echinocandins is challenging as the morphological differences are subtle. METHODS: The MECs of 10 WT and 4 non-WT Aspergillus fumigatus isolates were determined with the EUCAST E.Def 9.4. Plates were inoculated with increasing inocula (102-105 cfu/mL) and after 24 and 48 h of incubation, MECs were determined macroscopically (magnifying mirror) and microscopically (inverted microscope) by two observers, spectrophotometrically (OD at 405 nm) and colorimetrically (absorbance at 450/630 nm after 2 h incubation with 400 mg/L XTT/6.25 µM menadione). The interobserver (between observers)/intermethod (compared with the microscopic method) essential agreement (EA, ±1 2-fold dilution) and categorical agreement (CA) were determined for each inoculum. RESULTS: Echinocandin-induced microscopic hyphal alterations or macroscopic changes in turbidity were subtle with a 105 cfu/mL inoculum compared with the lower inocula of 103 and 102 cfu/mL, where more distinct changes in turbidity and formation of characteristic rosettes were obvious at the MEC after 48 h. A 105 cfu/mL inoculum resulted in wider MEC distributions (3-6 dilutions) and lower interobserver EA (69%), macroscopic-microscopic EA (26%) and CA (71%) compared with a 103 cfu/mL inoculum (2-3 dilutions, 100%, 100% and 100%, respectively). Spectrophotometric readings using a 103 cfu/mL inoculum showed good EA (57-93%) and excellent CA (86%-100%), while the XTT assay demonstrated excellent EA (93%) and CA (100%). CONCLUSIONS: A 48 h incubation using a 103 cfu/mL inoculum improved echinocandin MEC determination for A. fumigatus with the EUCAST method, while the colorimetric assay could allow automation.

Multicenter Evaluation of the BIOFIRE Blood Culture Identification 2 Panel for Detection of Bacteria, Yeasts, and Antimicrobial Resistance Genes in Positive Blood Culture Samples.

Diagnostic tools that can rapidly identify and characterize microbes growing in blood cultures are important components of clinical microbiology practice because they help to provide timely information that can be used to optimize patient management. This publication describes the bioMérieux BIOFIRE Blood Culture Identification 2 (BCID2) Panel clinical study that was submitted to the U.S. Food & Drug Administration. Results obtained with the BIOFIRE BCID2 Panel were compared to standard-of-care (SoC) results, sequencing results, PCR results, and reference laboratory antimicrobial susceptibility testing results to evaluate the accuracy of its performance. Results for 1,093 retrospectively and prospectively collected positive blood culture samples were initially enrolled, and 1,074 samples met the study criteria and were included in the final analyses. The BIOFIRE BCID2 Panel demonstrated an overall sensitivity of 98.9% (1,712/1,731) and an overall specificity of 99.6% (33,592/33,711) for Gram-positive bacteria, Gram-negative bacteria and yeast targets which the panel is designed to detect. One hundred eighteen off-panel organisms, which the BIOFIRE BCID2 Panel is not designed to detect, were identified by SoC in 10.6% (114/1,074) of samples. The BIOFIRE BCID2 Panel also demonstrated an overall positive percent agreement (PPA) of 97.9% (325/332) and an overall negative percent agreement (NPA) of 99.9% (2,465/2,767) for antimicrobial resistance determinants which the panel is designed to detect. The presence or absence of resistance markers in Enterobacterales correlated closely with phenotypic susceptibility and resistance. We conclude that the BIOFIRE BCID2 Panel produced accurate results in this clinical trial.

In Vitro-In Vivo Correlation of Posaconazole-Amphotericin B Combination against Candida albicans: In Vitro Interacting Concentrations Are Associated with In Vivo Free Drug Levels.

The in vitro/in vivo correlation of antifungal combination testing is necessary in order to assess the efficacy of combination regimens. We, therefore, attempted to correlate in vitro chequerboard testing of posaconazole (POS) and amphotericin B (AMB) with the in vivo outcome of combination therapy against experimental candidiasis in a neutropenic murine model. The AMB + POS combination was tested against a Candida albicans isolate. In vitro, a broth microdilution 8 × 12 chequerboard method with serial two-fold drug dilutions was used. In vivo, CD1 female neutropenic mice with experimental disseminated candidiasis were treated with i.p. AMB and p.o. POS alone and in combination at three effective doses (ED20, ED50 and ED80 corresponding to 20%, 50% and 80% of maximal effect, respectively). CFU/kidneys after 2 days were determined. The pharmacodynamic interactions were assessed based on Bliss independence interaction analysis. In vitro, a Bliss antagonism of -23% (-23% to -22%) was observed at 0.03-0.125 mg/L of AMB with 0.004-0.015 mg/L of POS, while a Bliss synergy of 27% (14%-58%) was observed at 0.008-0.03 mg/L of AMB with 0.000015-0.001 mg/L of POS. In vivo, Bliss synergy (13 ± 4%) was found when an AMB ED20 of 1 mg/kg was combined with all POS ED 0.2-0.9 mg/kg, while Bliss antagonism (35-83%) was found for the combinations of AMB ED50 2 mg/kg and ED80 3.2 mg/kg with POS ED80 of 0.9 mg/kg. Free drug serum levels of POS and AMB in in vivo synergistic and antagonistic combinations were correlated with the in vitro synergistic and antagonistic concentrations, respectively. Both synergistic and antagonistic interactions were found for the AMB + POS combination. POS compromised the efficacy of high effective AMB doses and enhanced low ineffective AMB doses. In vitro concentration-dependent interactions were correlated with in vivo dose-dependent interactions of the AMB + POS combination. In vivo interactions occurred at free drug serum levels close to in vitro interacting concentrations.

Overestimation of Amphotericin B Resistance in Candida auris with Sensititre YeastOne Antifungal Susceptibility Testing: a Need for Adjustment for Correct Interpretation.

Significant variation in minimal inhibitory concentrations (MIC) has been reported for amphotericin B (AMB) and C. auris, depending on the antifungal susceptibility testing (AFST) method. Although the Sensititre YeastOne (SYO) is widely used in routine laboratory testing, data regarding its performance for the AFST of C. auris are scarce. We tested AMB against 65 C. auris clinical isolates with the SYO and the reference methodology by the Clinical and Laboratory Standards Institute (CLSI). The essential agreement (EA, ±1 dilution) between the two methods and the categorical agreement (CA) based on the Centers for Disease Control and Prevention (CDC)'s tentative breakpoint of MIC ≥ 2 mg/L were determined. The SYO wild type upper limit value (WT-UL) was determined using the ECOFFinder. The modal (range) CLSI growth inhibitory MIC was lower than the SYO colorimetric MIC [1(0.25-1) versus 2(1-8) mg/L, respectively]). The CLSI-colorimetric SYO EA was 29% and the CA was 11% (89% major errors; MaE). MaE were reduced when the SYO WT-UL of 8 mg/L was used (0% MaE). Alternatively, the use of SYO growth inhibition endpoints of MIC-1 (75% growth inhibition) or MIC-2 (50% growth inhibition) resulted in 88% CA with 12% MaE and 97% CA with 3% MaE, respectively. In conclusion, SYO overestimated AMB resistance in C. auris isolates when colorimetric MICs, as per SYO instructions and the CDC breakpoint of 2 mg/L, were used. This can be improved either by using the method-specific WT-UL MIC of 8 mg/L for colorimetric MICs or by determining growth inhibition MIC endpoints, regardless of the color. IMPORTANCE Candida auris is an emerging and frequently multidrug-resistant fungal pathogen that accounts for life-threatening invasive infections and nosocomial outbreaks worldwide. Reliable AF is important for the choice of the optimal treatment. Commercial methods are frequently used without prior vigorous assessment. Resistance to AMB was over-reported with the commercial colorimetric method Sensititre YeastOne (SYO). SYO produced MICs that were 1 to 2 twofold dilutions higher than those of the reference CLSI method, resulting in 89% MaE. MaE were reduced using a SYO-specific colorimetric wild type upper limit MIC value of 8 mg/L (0%) or a 50% growth inhibition endpoint (3%).

Assessing Clinical Potential of Old Antibiotics against Severe Infections by Multi-Drug-Resistant Gram-Negative Bacteria Using In Silico Modelling.

In the light of increasing antimicrobial resistance among gram-negative bacteria and the lack of new more potent antimicrobial agents, new strategies have been explored. Old antibiotics, such as colistin, temocillin, fosfomycin, mecillinam, nitrofurantoin, minocycline, and chloramphenicol, have attracted the attention since they often exhibit in vitro activity against multi-drug-resistant (MDR) gram-negative bacteria, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. The current review provides a summary of the in vitro activity, pharmacokinetics and PK/PD characteristics of old antibiotics. In silico modelling was then performed using Monte Carlo simulation in order to combine all preclinical data with human pharmacokinetics and determine the probability of target (1-log kill in thigh/lung infection animal models) attainment (PTA) of different dosing regimens. The potential of clinical efficacy of a drug against severe infections by MDR gram-negative bacteria was considered when PTA was >95% at the epidemiological cutoff values of corresponding species. In vitro potent activity against MDR gram-negative pathogens has been shown for colistin, polymyxin B, temocillin (against E. coli and K. pneumoniae), fosfomycin (against E. coli), mecillinam (against E. coli), minocycline (against E. coli, K. pneumoniae, A. baumannii), and chloramphenicol (against E. coli) with ECOFF or MIC90 ≤ 16 mg/L. When preclinical PK/PD targets were combined with human pharmacokinetics, Monte Carlo analysis showed that among the old antibiotics analyzed, there is clinical potential for polymyxin B against E. coli, K. pneumoniae, and A. baumannii; for temocillin against K. pneumoniae and E. coli; for fosfomycin against E. coli and K. pneumoniae; and for mecillinam against E. coli. Clinical studies are needed to verify the potential of those antibiotics to effectively treat infections by multi-drug resistant gram-negative bacteria.

Can bronchial secretion cultures identify the etiologic agent of COVID-19-associated pulmonary aspergillosis in ICU patients? Comparison with a species-specific Aspergillus PCR in serum.

Aspergillus spp. isolated from non-BAL cultures of coronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) patients may reflect colonization rather than infection. Sera (n = 181) from 49 adult ICU CAPA patients (24 probable and 25 possible CAPA) with bronchial secretions (BS) culture positive for Aspergillus spp. were collected and tested for Aspergillus DNA detection by species-specific real-time PCR. Overall, 30/49 (61%) patients were PCR positive. BS culture/serum PCR agreement was moderate (21/30; 70%). Based on serum PCR positive patients, all CAPAs were due to A. fumigatus (80%), A. flavus (10%), and A. terreus (10%). No A. niger/A. nidulans or mixed infections were found despite positive BS cultures.

Discordant results were observed between bronchial secretion cultures and species-specific serum PCR (30%) with A. fumigatus being by far the most common etiological agent of CAPA (80%). No A. niger/A. nidulans or mixed infections were found despite positive cultures.