Single dose pharmacodynamics of amphotericin B against Aspergillus species in an in vitro pharmacokinetic/pharmacodynamic model

Conventional MIC testing of amphotericin B results in narrow MIC ranges challenging the detection of resistant strains. In order to discern amphotericin B pharmacodynamics, the in vitro activity of amphotericin B was studied against Aspergillus isolates with the same MIC with a new in vitro pharmacokinetic/pharmacodynamic (PK/PD) model that simulates amphotericin B human plasma levels. Clinical isolates of A. fumigatus, A. terreus and A flavus with the same CLSI modal MICs of 1 mg/l were exposed to amphotericin B concentrations following the plasma concentration-time profile after single bolus administration with Cmax 0.6, 1.2, 2.4 and 4.8 mg/L. Fungal growth was monitored up to 72h based on galactomannan production. Complete growth inhibition was observed only against A. fumigatus with amphotericin B Cmax ≥2.4 mg/L. At lower Cmaxs 0.6 and 1.2 mg/L, a significant growth delay of 34h and 52h was observed, respectively (pA flavus>A. terreus in the in vitro PK/PD model possibly reflecting the different concentration- and time-dependent inhibitory/killing activities amphotericin B exerting against these species

The in vitro and in vivo testing of chemotherapeutic agents against pathogenic free living amebae : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand.

During the last ten years, there has been an increasing awareness of sporadic cases of Primary Amoebic Meningo-encephalitis (PAM) affecting primarily younger age groups and appearing in an acute fulminant form. The earliest positive case (Willaert, 1974) may have been in England in 1909 which shows that the disease has been with us for a long time. The pathogenic free-living amebae (PFLA), which comprises the genus Naegleria and the genus Acanthamoeba, are the causative organisms of PAM and AM*respectively. PAM is a rapidly fatal disease affecting the central nervous system (CNS),the treatment of which to date has been successful in only a small number of cases, and therefore the continual screening of suitable chemotherapeutic agents against amebae of the Naegleria spp. and Acanthamoeba spp., is of great importance. AM is also essentially confined to the CNS although it may take the form of chronic granulomata in the liver, spleen, uterus and kidneys (Martinez et al., 1977). Six chemotherapeutic agents: Amphotericin B, 5-Fluorocytosine, Kanamycin, Oxytetracycline, Tylosine and Levamisole were tested for activity against a non-pathogenic and a pathogenic species of Naegleria and a non-pathogenic and a pathogenic species of Acanthamoeba in axenic culture. For the Naegleria spp., Amphotericin B and Oxytetracycline were found to be active and the Acanthamoeba spp. were found to be only susceptible to Levamisole. The synergistic combinations of drugs against the amebae were also investigated in axenic culture. In preliminary trials Kanamycin together with Oxytetracycline showed promise against Naegleria fowleri (MsM) but this was later shown not to be the case. Amphotericin B in combination with 5-Fluorocytosine was also shown not to be synergistic, however Amphotericin B in combination with Oxytetracycline proved to be effective against N. fowleri. Amphotericin B was combined with 5-Fluorocytosine against A. culbertsoni (A-1) but was not found to be synergistically active. * Amebic meningitis caused by Acanthamoeba infections. Levamisole was also tested against N. gruberi (P1200f) and A. castellanii (0.1) at various stages in growth of the amebae (i.e. 24, 48 and 72 hour stock cultures) to determine the effect of using aged amebae. It was found that the age of the stock culture bore no relation to the activity of the drug. After axenic culture testing, the susceptibility of the pathogenic N. fowleri (MsM) and A. culbertsoni (A-1) to the agents which showed activity, was investigated in a vero cell culture system. For N. fowleri (MsM) the results of axenic culture testing were confirmed, with Amphotericin B and Oxytetracycline protecting the monolayer from the destructive effects of the amebae, both when used singly and at a greater efficiency when added together as a synergistic combination. Levamisole, although effective to some extent against Acanthamoeba spp. in axenic culture, failed to show any activity against the amebae in vero cell culture testing. In vivo animal protection studies were then performed using drugs that had been shown either in this or other studies to be effective against either Naegleria or Acanthamoeba spp. Chemotherapeutic agents tested on N. fowleri (MsM) included two imidazoles; Miconazole nitrate and Ketoconazole (previously known as R41,400), as well as Amphotericin B. The synergistic combination of Amphotericin B with either Tetracycline or Oxytetracycline was also investigated. For A. culbertsoni (A-1), 5-Fluorocytosine, and Polymyxin B were tried both singly and in combination. These drugs were injected by intraperitoneal (I.P.) and intraventricular (I.vent.) routes. The results were not promising, with none of the drugs offering significant protection even whilst using Amphotericin B which is considered the drug of choice. The question of adequate drug levels reaching the brain was tested out with two imidazoles, Ketoconazole and Miconazole. Serum samples were assayed against Candida pirapsilosis and C. pseudotropicalis respectively at various time intervals after innoculation with the drug, and a gradual increase and breakdown of the drug in the animal system could then be shown. These results showed that based on in vitro results, the levels of the imidazoles obtained in the serum after the first eight hours after injection, should have been sufficiently high to prevent amebic multiplication

Limited effectiveness of high-dose liposomal amphotericin B (AmBisome) for treatment of visceral leishmaniasis in an Ethiopian population with high HIV prevalence.

Due to unacceptably high mortality with pentavalent antimonials, Médecins Sans Frontières in 2006 began using liposomal amphotericin B (AmBisome) for visceral leishmaniasis (VL) patients in Ethiopia who were severely ill or positive for human immunodeficiency virus (HIV)

Amphotericin forms an extramembranous and fungicidal sterol sponge.

For over 50 years, amphotericin has remained the powerful but highly toxic last line of defense in treating life-threatening fungal infections in humans with minimal development of microbial resistance. Understanding how this small molecule kills yeast is thus critical for guiding development of derivatives with an improved therapeutic index and other resistance-refractory antimicrobial agents. In the widely accepted ion channel model for its mechanism of cytocidal action, amphotericin forms aggregates inside lipid bilayers that permeabilize and kill cells. In contrast, we report that amphotericin exists primarily in the form of large, extramembranous aggregates that kill yeast by extracting ergosterol from lipid bilayers. These findings reveal that extraction of a polyfunctional lipid underlies the resistance-refractory antimicrobial action of amphotericin and suggests a roadmap for separating its cytocidal and membrane-permeabilizing activities. This new mechanistic understanding is also guiding development of what are to our knowledge the first derivatives of amphotericin that kill yeast but not human cells

Treatment of Primary Pulmonary Aspergillosis: An Assessment of the Evidence.

Aspergillus spp. are a group of filamentous molds that were first described due to a perceived similarity to an aspergillum, or liturgical device used to sprinkle holy water, when viewed under a microscope. Although commonly inhaled due to their ubiquitous nature within the environment, an invasive fungal infection (IFI) is a rare outcome that is often reserved for those patients who are immunocompromised. Given the potential for significant morbidity and mortality within this patient population from IFI due to Aspergillus spp., along with the rise in the use of therapies that confer immunosuppression, there is an increasing need for appropriate initial clinical suspicion leading to accurate diagnosis and effective treatment. Voriconazole remains the first line agent for therapy; however, the use of polyenes, novel triazole agents, or voriconazole in combination with an echinocandin may also be utilized. Consideration as to which particular agent and for what duration should be made in the individual context for each patient based upon underlying immunosuppression, comorbidities, and overall tolerance of therapy

Biofilm-forming capability of highly virulent, multidrug-resistant Candida auris

The emerging multidrug-resistant yeast pathogen Candida auris has attracted considerable attention as a source of healthcare–associated infections. We report that this highly virulent yeast has the capacity to form antifungal resistant biofilms sensitive to the disinfectant chlorhexidine in vitro

Bloodstream yeast infections: a 15-month survey

A 15-month survey of 412 bloodstream yeast isolates from 54 Belgian hospitals was undertaken. Candida albicans was the most common species (47.3%) followed by C. glabrata (25.7%), C. parapsilosis (8.0%), C. tropicalis (6.8%) and Saccharomyces cerevisiae (5.1%). Common predisposing factors were antibacterial therapy (45%), hospitalization in intensive care units (34%), presence of in-dwelling catheters (32%), underlying cancer (23%) and major surgery (11%). Most patients had more than one predisposing factor. Fluconazole alone or in combination with another antifungal agent was the treatment of choice for 86.6% of the cases. Susceptibility testing revealed that 93.5% were susceptible to amphotericin B, 39.6% to itraconazole, 42.8% to fluconazole and 87% to voriconazole. Resistance to azoles was more common among C. glabrata isolates

Absent in vitro interaction between chloroquine and antifungals against Aspergillus fumigatus

This work was supported by Medical Mycology Section, Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, the Netherlands. Parts of these results were presented at the ASM Microbe Conference of American Society for Microbiology, June 16–20, 2016, Boston, MA, Poster no. 426,Peer reviewedPostprin