Precision Therapy for Invasive Fungal Diseases

Invasive fungal infections (IFI) are a common infection-related cause of death in immunocompromised patients. Approximately 10 million people are at risk of developing invasive aspergillosis annually. Detailed study of the pharmacokinetics (PK) and pharmacodynamics (PD) of antifungal drugs has resulted in a better understanding of optimal regimens for populations, drug exposure targets for therapeutic drug monitoring, and establishing in vitro susceptibility breakpoints. Importantly, however, each is an example of a “one size fits all strategy”, where complex systems are reduced to a singularity that ensures antifungal therapy is administered safely and effectively at the level of a population. Clearly, such a notion serves most patients adequately but is completely counter to the covenant at the centre of the clinician–patient relationship, where each patient should know whether they are well-positioned to maximally benefit from an antifungal drug. This review discusses the current therapy of fungal infections and areas of future research to maximise the effectiveness of antifungal therapy at an individual level.

In Vitro Susceptibility of Talaromyces Marneffei In Malaysia: Comparison of Yeast and Mycelial Phases

Talaromyces marneffei is an etiologic agent of talaromycosis. It can cause serious complications and death in immunocompromised patients, particularly in acquired immunodeficiency syndrome (AIDS) patients. This infectious disease is endemic in Southeast Asia including Malaysia. To date, published reports on the antifungal susceptibility profile of T. marneffei is very limited. The objective of this study is to determine the minimum inhibitory concentration (MIC) of T. marneffei in yeast and mycelial phases in Malaysia. In the year 2020, 27 clinical strains of T. marneffei were received from various hospitals in Malaysia. The identification was carried out using microscopic, macroscopic and molecular methods. Following that, the susceptibility of each isolate in both yeast and mycelial form to thirteen common antifungals was performed according to the broth microdilution in Clinical & Laboratory Standards Institute (CLSI) M38 method. The antifungals tested were anidulafungin, micafungin sodium, caspofungin diacetate, 5-fluorocytosine, amphotericin B and terbinafine hydrochloride, posaconazole, voriconazole, itraconazole, ketoconazole, ravuconazole, clotrimazole and isavuconazole. The geometric mean of all antifungals other than anidulafungin, micafungin sodium, caspofungin diacetate and 5-fluorocytosine against T. marneffei mould (mycelial) were >2 μg/ml. However, the geometric mean of all antifungals against T. marneffei yeast was <2 μg/ml. Our in vitro data suggests promising activities of amphotericin B, terbinafine hydrochloride, posaconazole, voriconazole, itraconazole, ketoconazole, ravuconazole, clotrimazole and isavuconazole against yeast and mould phases of T. marneffei.

Precise editing using CRISPR-Cas9 to explore the contribution of clinically-derived mutations to antifungal resistance in the pathogenic yeast Candida parapsilosis

Introduction Candida parapsilosis is both a commensal/saprophytic yeast of the human skin and an opportunistic pathogen which can be responsible for life-threatening infections. The increasing reports of clonal outbreaks involving azole-resistant C. parapsilosis in the clinical setting is worrisome and urges for a better understanding of antifungal resistance in this species. Previous studies have identified mutations in key genes which can explain acquired fluconazole resistance. Reverse genetics approaches are now warranted to confirm their involvement and to determine whether they can affect other clinically-licensed antifungals. Here, we used a CRISPR-Cas9 technique to study the relative contributions of clinically-derived mutations to antifungal resistance and provide answers to these questions. Materials and Methods Six clinically-derived mutations were selected (ERG11Y132F, ERG11K143R,ERG11R398I, TAC1G650E, MRR1G583R, ERG3G111R) to be engineered in two C. parapsilosis fluconazole-susceptible backgrounds (ATCC22019, STZ5) using a previously described CRISPR-Cas9 method. In vitro susceptibility of the transformants to fluconazole, voriconazole, posaconazole, isavuconazole and micafungin was determined by Etest®. Results/Discussion The impact on fluconazole susceptibility was highly variable depending on the residue/gene involved, but roughly similar between the two genetic backgrounds. All but two(ERG11R398I, ERG3G111R) conferred fluconazole resistance, though the highest MIC increase was observed for MRR1G583R (≥650 fold). As expected in a diploid species, we noted an impact of allelic dosage. Some kind of cross-resistance to the other azoles was noted from some mutations, although the impact was lower for posaconazole and isavuconazole, except for MRR1G583R which led to multi-azole resistance. Finally, ERG3G111R increased tolerance to both azoles and echinocandins.

Increased Leishmania infantum resistance to miltefosine and amphotericin B after treatment of a dog with miltefosine and allopurinol

Background Leishmania infantum is the most important etiological agent of visceral leishmaniasis in the Americas and Mediterranean region, and the dog is the main host. Miltefosine was authorized to treat canine leishmaniasis (CanL) in Brazil in 2017, but there is a persistent fear of the emergence of parasites resistant not only to this drug but, through cross-resistance mechanisms, also to meglumine antimoniate and amphotericin B. Additionally, the literature shows that acquisition of resistance is followed by increased parasite fitness, with higher rates of proliferation, infectivity and metacyclogenesis, which are drivers of parasite virulence. In this context, the aim of this study was to analyze the impact of treating a dog with miltefosine and allopurinol on the generation of parasites resistant to miltefosine, amphotericin B and meglumine antimoniate. Methods In vitro susceptibility tests were conducted against miltefosine, amphotericin B and meglumine antimoniate with T0 (parasites isolated from a dog before treatment with miltefosine plus allopurinol), T1 (after 1 course of treatment) and T2 (after 2 courses of treatment) isolates. The rates of cell proliferation, infectivity and metacyclogenesis of the isolates were also evaluated. Results The results indicate a gradual increase in parasite resistance to miltefosine and amphotericin B with increasing the number of treatment courses. An increasing trend in the metacyclogenesis rate of the parasites was also observed as drug resistance increased. Conclusion The data indicates an increased L. infantum resistance to miltefosine and amphotericin B after the treatment of a dog with miltefosine plus allopurinol. Further studies with a larger number of L. infantum strains isolated from dogs with varied immune response profiles and undergoing different treatment regimes, are advocated. Graphical Abstract

In Vitro Susceptibility of Gram-negative Pathogens to Cefiderocol in Five Consecutive Annual Multinational SIDERO-WT Surveillance Studies (2014-2019)

We report in vitro susceptibility data from five consecutive annual SIDERO-WT surveillance studies (2014-2019) for cefiderocol and comparators tested against Gram-negative clinical isolates from North America and Europe. CLSI broth microdilution was used to determine MICs for Enterobacterales ( n =31,896), Pseudomonas aeruginosa ( n =7,700), Acinetobacter baumannii complex ( n =5,225), Stenotrophomonas maltophilia ( n =2,030), and Burkholderia cepacia complex ( n =425). MICs were interpreted by CLSI-approved clinical breakpoints (February 2021). Cefiderocol inhibited 99.8%, 96.7%, 91.6%, and 97.7% of all Enterobacterales , meropenem-nonsusceptible, ceftazidime-avibactam-nonsusceptible, and ceftolozane-tazobactam-nonsusceptible isolates, respectively, at ≤4 μg/ml (susceptible breakpoint). Cefiderocol inhibited 99.9%, 99.8%, 100%, and 99.8% of all P. aeruginosa , meropenem-nonsusceptible, ceftazidime-avibactam-nonsusceptible, and ceftolozane-tazobactam-nonsusceptible isolates, respectively, at ≤4 μg/ml (susceptible breakpoint). Cefiderocol inhibited 96.0% of all A. baumannii complex isolates and 94.2% of meropenem-nonsusceptible isolates at ≤4 μg/ml (susceptible breakpoint), and 98.6% of S. maltophilia isolates at ≤1 μg/ml (susceptible breakpoint). B. cepacia complex isolates tested with a MIC 50 of ≤0.03 μg/ml and MIC 90 of 0.5 μg/ml. Annual cefiderocol percent susceptible rates for Enterobacterales (North America, range 99.6-100%/year; Europe, range 99.3-99.9%/year) and P. aeruginosa (99.8-100%; 99.9-100%) were unchanged from 2014 to 2019. Annual percent susceptible rates for A. baumannii complex demonstrated sporadic, non-directional differences (97.5-100%; 90.4-97.5%); the wider range for Europe (∼7%) was due to isolates from Russia. Annual percent susceptible rates for S. maltophilia showed minor, non-directional differences (96.4-100%; 95.6-100%). We conclude that clinical isolates of Enterobacterales (99.8% susceptible), P. aeruginosa (99.9%), A. baumannii (96.0%), and S. maltophilia (98.6%) collected in North America and Europe from 2014 to 2019 were highly susceptible to cefiderocol.

Impact of FKS 1 genotype on echinocandin in-vitro susceptibility in Candida auris and in -vivo response in a murine model of infection

Objectives: Echinocandins are frontline antifungal agents in the management of invasive infections due to multi-drug resistant Candida auris . The study aimed to evaluate echinocandin resistance in C. auris isolates of multicentric origin, identify the resistance mechanism, and analyze the pharmacodynamic response to caspofungin in a neutropenic mouse model of infection. Methods : A total of 199 C. auris isolates originating from thirty centres across India were tested for susceptibility to echinocandins. Isolates with reduced susceptibility were evaluated for FKS 1 mutations and in-vivo response to caspofungin in a murine model of disseminated candidiasis. In addition, the response to echinocandins was assessed in light of in-vitro growth kinetics, chitin content; and transcript levels of chitin synthase and FKS1 genes. Results: We report 10 resistant C. auris isolates with four FKS 1 mutations: F635Y ( n =2), F635L ( n =4), S639F ( n =3), and R1354S ( n =1). Of these, F635Y and R1354S exhibited the most profound resistance in mouse model of disseminated infection. S639F and F635L mutations conferred a moderate in vivo resistance, whereas wild-type isolates exhibiting borderline MIC were susceptible in vivo . FKS 1 genotype was more accurate predictor of in-vivo response than the MIC of the isolates. Isolates with high basal or inducible chitin content exhibited higher in vitro MIC in FKS 1 mutant compared to wild-type. Conclusions FKS 1 mutations play a major role in clinically relevant echinocandin resistance in C. auris with differential in vivo outcomes. This study could have implications for clinical practice and, therefore, warrants further studies.

Population Pharmacokinetics of Meropenem in Critically Ill Korean Patients and Effects of Extracorporeal Membrane Oxygenation

Limited studies have investigated population pharmacokinetic (PK) models and optimal dosage regimens of meropenem for critically ill adult patients using the probability of target attainment, including patients receiving extracorporeal membrane oxygenation (ECMO). A population PK analysis was conducted using non-linear mixed-effect modeling. Monte Carlo simulation was used to determine for how long the free drug concentration was above the minimum inhibitory concentration (MIC) at steady state conditions in patients with various degrees of renal function. Meropenem PK in critically ill patients was described using a two-compartment model, in which glomerular filtration rate was identified as a covariate for clearance. ECMO did not affect meropenem PK. The simulation results showed that the current meropenem dosing regimen would be sufficient for attaining 40%fT>MIC for Pseudomonas aeruginosa at MIC ≤ 4 mg/L. Prolonged infusion over 3 h or a high-dosage regimen of 2 g/8 h was needed for MIC > 2 mg/L or in patients with augmented renal clearance, for a target of 100%fT>MIC or 100%fT>4XMIC. Our study suggests that clinicians should consider prolonged infusion or a high-dosage regimen of meropenem, particularly when treating critically ill patients with augmented renal clearance or those infected with pathogens with decreased in vitro susceptibility, regardless of ECMO support.

LB16. Surveillance Rates of Cefiderocol Heteroresistance Correlate With All-cause Mortality in the APEKS-NP and CREDIBLE-CR Trials

Background Cefiderocol is a recently FDA approved, novel siderophore beta-lactam antibiotic. Conventional antimicrobial susceptibility testing (AST) suggests that a variety of Gram-negative pathogens, including carbapenem-resistant (CR) isolates, are overwhelmingly susceptible to cefiderocol.. This antibiotic performed well in the APEKS-NP trial for the treatment of nosocomial pneumonia caused largely by carbapenem susceptible isolates. However, in the CREDIBLE-CR trial involving exclusively CR Gram-negative bacteria, cefiderocol was associated with a higher rate of all-cause mortality. We hypothesized one explanation for these discrepant data might be undetected cefiderocol heteroresistance (HR). HR is a form of antibiotic resistance in which an isolate harbors a minority resistant subpopulation of cells co-existing with a majority susceptible population, and is often undetected by standard AST. Isolates exhibiting undetected HR, with as low as 1 in 1 million resistant cells, can cause treatment failure in in vivo models. Methods We quantified HR to cefiderocol by population analysis profile (PAP) of 161 Acinetobacter, 180 Klebsiella, and 108 Pseudomonas isolates collected in Georgia, USA. Results We observed CR isolates exhibited a high frequency of HR, which was largely undetected by standard AST, and correlated with all-cause mortality in the CREDIBLE-CR study (Table). Carbapenem-susceptible isolates exhibited no or low rates of cefiderocol HR (Table). Cephalosporin-resistant bacteria mostly exhibited increased rates of cefiderocol HR, but below those of CR strains. These differences in rates of cefiderocol HR correlated with the mortality data from the APEKS-NP and CREDIBLE-CR trials, across the bacterial species tested (Table). Table: Surveillance rates of cefiderocol heteroresistance correlate with all-cause mortality in the APEKS-NP and CREDIBLE-CR trials Conclusion These data suggest that the lower rates of cefiderocol HR in carbapenem-susceptible isolates that predominated the APEKS-NP trial may explain the enhanced efficacy of the drug in that study as compared to the CREDIBLE-CR trial. Importantly, the widespread, undetected cefiderocol HR observed among CR pathogens may explain the discordance between this drug’s excellent in vitro susceptibility profile and increased patient mortality in the CREDIBLE-CR trial. Disclosures All Authors: No reported disclosures