Wim van Vianen

In Vitro Susceptibilities of Madurella mycetomatis to Itraconazole and Amphotericin B Assessed by a Modified NCCLS Method and a Viability-Based 2,3-Bis(2-Methoxy-4-Nitro-5- Sulfophenyl)-5-[(Phenylamino)Carbonyl]-2H- Tetrazolium Hydroxide (XTT) Assay

ABSTRACT Susceptibilities of Madurella mycetomatis against amphotericin B and itraconazole in vitro were determined by protocols based on NCCLS guidelines (visual reading) and a 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) assay for fungal viability. The XTT assay was reproducible and sensitive for both antifungals. Itraconazole (MIC at which 50% of the isolates tested are inhibited [MIC50]) of 0.06 to 0.13 mg/liter) was superior to amphotericin B (MIC50 of 0.5 to 1.0 mg/liter).

Isolation and characterization of murine Kupffer cells and splenic macrophages

A method is described using counterflow centrifugation elutriation to isolate macrophages from murine liver and spleen. In this study three, size fractionated, macrophage populations were collected. Isolation resulted in a high yield of pure Kupffer cells (total of 10 x 10(6) /g liver) and enrichment of splenic macrophages to 20%. In addition to standard methods such as non-specific esterase staining, the isolated macrophages were also characterized by flow cytometry using specific monoclonal antibodies. In addition, a rapid flow cytometry method was introduced to determine the percentage of macrophages based on autofluorescence. A strong correlation was found between the percentages of macrophages found by non-specific esterase staining and autofluorescence. Functional tests revealed differences between the isolated macrophages in terms of tumor necrosis factor-alpha (TNF-alpha) production, oxygen metabolism and the production of nitric oxide. However, no significant differences in phagocytic activity was observed between the fractions. After two weeks of culture without the addition of antibiotics the cells still exhibited the above mentioned functions.

Combination therapy of advanced invasive pulmonary aspergillosis in transiently neutropenic rats using human pharmacokinetic equivalent doses of voriconazole and anidulafungin

ABSTRACT At present, voriconazole (VOR) is the drug of first choice for treating invasive pulmonary aspergillosis (IPA). However, particularly in advanced stages of disease and in the severely immunocompromised host, the mortality remains substantial. The combination of VOR with an echinocandin may improve the therapeutic outcome. We investigate here whether combining VOR and anidulafungin (ANI) in advanced IPA in transiently neutropenic rats results in a higher therapeutic efficacy. Since VOR is metabolized more rapidly in rodents than in humans, dosage adjustment for VOR is necessary to obtain an area under the plasma concentration-time curve (AUC) in rodents that is equivalent to that of humans. In this study, the pharmacokinetics of VOR and ANI in rats were elucidated, and dosage schedules were applied that produced AUCs similar to those of humans. The developed dose schedules were well tolerated by the rats, without effects on renal and hepatic functions. VOR showed excellent efficacy in early IPA (100% rat survival). In advanced IPA, VOR was less efficacious (50% rat survival), whereas a significant decrease in galactomannan concentrations in lungs and sera was found in surviving rats. ANI administered in advanced IPA resulted in 22% rat survival, and the serum concentrations of fungal galactomannan were slightly but not significantly decreased. The addition of ANI to VOR did not result in significantly increased therapeutic efficacy in advanced IPA, resulting in 67% rat survival and a significant decrease in galactomannan concentration in serum. In conclusion, VOR monotherapy is therapeutically effective in the treatment of advanced-stage IPA and superior to the use of ANI. Combining both agents does not significantly improve the therapeutic outcome.

A murine model of Madurella mycetomatis eumycetoma

Eumycetoma due to Madurella mycetomatis is a major mycological health problem in endemic areas. We infected BALB/c mice (male or female) with various amounts of M. mycetomatis mycelium, containing sterilized soil as a natural adjuvant or Freunds incomplete adjuvant. Mice differed with respect to age and immune status. Intraperitoneal, subcutaneous and intravenous inoculation was explored and survival was monitored. Mice were killed at various intervals after inoculation, checked for the presence of the characteristic black grains, and organs were cultured for M. mycetomatis. Infected organs were subjected to histopathological examination. Immunocompetent male mice were as susceptible as immunocompromised female mice, but showed higher mortality rates. In conclusion, a reproducible mouse model of intraperitoneal M. mycetomatis infection with characteristic black grains in immunocompetent adult or young female mice was developed. Although this experimental model does not simulate macroscopic features of the subcutaneous M. mycetomatis infection in humans, the histopathological characteristics of the lesions and the development of black grains are clearly representative for the human infection. This model will enable further studies on the pathogenesis as well as prevention and treatment of the fungal infection.

Sterically stabilized amphotericin B-liposomes: Toxicity and biodistribution in mice

In this study it was investigated whether long-circulating amphotericin B (AMB) containing liposomes could be prepared by incorporation of polyethylene glycol (1900) derivatized distearoylphosphatidylethanolamine (PEG-DSPE), and whether the incorporation of PEG-DSPE affected toxicity and biodistribution of the preparation in mice. Toxicity of two formulations of liposomes containing both PEG-DSPE and AMB (PEG-AMB-LIP, types 1 and 2) was compared with that of AMB-liposomes without PEG-DSPE (AMB-LIP) as well as that of MB-deoxycholate (AMB-DOC). The maximum tolerated dosage (MTD) of AMB-DOC, expressed in terms of death during treatment for 5 consecutive days or significant increases in the parameters used to monitor renal and hepatic functions, was 0.8 mg/kg per day. AMB-LIP were the least toxic, the MTD being 11 mg/kg per day. The formulation with AMB complexed to DSPG (PEG-AMB-LIP type 1) was as toxic as AMB-DOC. This PEG-AMB-LIP formulation was omitted from further studies on biodistribution. With AMB complexed to PEG-DSPE (PEG-AMB-LIP type 2) toxicity was substantially reduced, resulting in a MTD of 9 mg/kg per day. Biodistribution of radiolabeled PEG-AMB-LIP type 2 was compared with that of AMB-LIP. Blood residence time of PEG-AMB-LIP type 2 was prolonged as compared to AMB-LIP; For PEG-AMB-LIP type 2 at 24 h after administration 30% of the injected dosage of AMB in intact liposomes was circulating versus 6% for AMB-LIP.

Antimycobacterial Agents Differ with Respect to Their Bacteriostatic versus Bactericidal Activities in Relation to Time of Exposure, Mycobacterial Growth Phase, and Their Use in Combination

ABSTRACT A number of antimycobacterial agents were evaluated with respect to their bacteriostatic activity (growth inhibition) versus the bactericidal activity against a clinical isolate of Mycobacterium avium (Mycobacterium avium complex [MAC] strain 101) in relation to the time of exposure and the growth phase of the mycobacteria. In terms of growth inhibition the MAC in the active phase of growth was susceptible to clarithromycin, ethambutol, rifampin, amikacin, and the quinolones moxifloxacin, ciprofloxacin, and sparfloxacin. In terms of bactericidal activity in relation to the time of exposure these agents differed substantially with respect to the killing rate. An initial high killing capacity at low concentration was observed for amikacin, which in this respect was superior to the other agents. The bactericidal activity of clarithromycin and ethambutol was only seen at relatively high concentrations and increased with time. Killing by rifampin was concentration dependent as well as time dependent. The bactericidal activity of moxifloxacin was marginally dependent on the concentration or the time of exposure. The activity of clarithromycin in combination with ethambutol was not significantly enhanced compared to single-agent exposure. Only an additive effect was observed. The addition of rifampin or moxifloxacin as a third agent only marginally effected increased killing of MAC. However, by addition of amikacin the activity of the clarithromycin-ethambutol combination was significantly improved. The combination of amikacin and amoxicillin-clavulanic acid exhibited synergistic antimycobacterial activity. Towards MAC at low growth rates, only the quinolones exhibited a bactericidal effect.

The effects of antifungal agents to conidial and hyphal forms of Aspergillus fumigatus.

Susceptibility testing for Aspergillus fumigatus is usually performed using a fungal conidial suspension. However, assessment of the susceptibility of fungal hyphae may be more relevant in attempting to mimic the fungal status in infected tissues. In the present study of 12 A. fumigatus clinical isolates and 1 ATCC strain, the antifungal susceptibilities of conidial suspensions, suspensions of hyphal fragments and of hyphal clumps were determined by the XTT-based broth susceptibility assay measuring decrease in fungal metabolic activity. Amphotericin B inhibited A. fumigatus conidia and hyphal fragments in a sharp concentration-dependent manner, with inhibitory concentrations (ICs) of 1 microg/ml for both fungal structures, whereas, hyphal clumps were inhibited at 8 microg/ml. Conidia and hyphal fragments were inhibited by the azoles itraconazole and voriconazole in a more gradual concentration-dependent manner, with ICs of 0.5 microg/ml for both structures with both agents. Hyphal clumps were not inhibited by the azoles at all. Caspofungin inhibited A. fumigatus in a moderate, neither sharp nor gradual, concentration-dependent manner. ICs for conidia were 128 microg/ml and inhibition in metabolic activity was not obtained for both hyphal growth forms. Antifungal susceptibility of conidia was also determined using the E-test in which it was found that the XTT assaygave comparable ICs for amphotericin B, itraconazole and voriconazole but not for caspofungin.

Caspofungin Prolongs Survival of Transiently Neutropenic Rats with Advanced-Stage Invasive Pulmonary Aspergillosis

ABSTRACT A high-dose-step-down strategy for caspofungin treatment was evaluated in an experimental model of advanced-stage invasive pulmonary aspergillosis. The therapeutic efficacy of caspofungin in relation to the severity of invasive pulmonary infection caused by Aspergillus fumigatus in transiently neutropenic rats was investigated by using rat survival and the decrease in the fungal burden as the parameters of efficacy. When treatment was started at either 16 h or 24 h after fungal inoculation, caspofungin administered intraperitoneally at 4 mg/kg of body weight/day for 10 days was highly effective (100% and 93% rat survival, respectively). However, only 27% rat survival was obtained when treatment was started at 72 h, when the rats had advanced-stage infection. Increasing the dose from 4 to 10 mg/kg/day could compensate for the decrease in efficacy and resulted in 67% rat survival. The high dose of 10 mg/kg/day for 10 days did not appear to be necessary since a high-dose-step-down dosing schedule with 10 mg/kg/day for 3 days followed by 4 mg/kg/day for 7 days was equally effective. At 10 days after the end of treatment with 10 mg/kg/day caspofungin, the level of neither A. fumigatus DNA nor A. fumigatus galactomannan in the infected left lung was significantly decreased. In contrast, A. fumigatus galactomannan concentrations in serum were significantly decreased. The levels of creatinine, blood urea nitrogen, alanine aminotransferase, and asparate aminotransferase were not elevated during treatment. Caspofungin is effective for the treatment of invasive pulmonary aspergillosis in transiently neutropenic rats and is even effective in rats with advanced-stage infection. In this model, the administration of high-dose-step-down treatment was as effective as treatment with high doses for the whole treatment period.

Amphotericin B but not itraconazole is able to prevent grain formation in experimental Madurella mycetomatis mycetoma in mice

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