The toxicology of liposome-encapsulated amphotericin B in mice and its efficacy in the treatment and prophylaxis of systemic candidiasis in these animals were studied. The toxicology studies indicated that the maximal tolerated dose of free amphotericin B was 0.8 mg/kg of body weight and the 50% lethal dose (LD50) was reached at 1.2 mg/kg, while neither the maximal tolerated dose nor the LD50 for the liposomal amphotericin B was reached at a dose of 12 mg/kg. No abnormalities in blood chemistry or histology were observed in the animals injected with encapsulated amphotericin B, while the administration of free amphotericin B was associated with nephrocalcinosis and renal parenchymal edema. The encapsulated drug was as effective as the free drug when used in similar concentrations, while the animals treated with higher concentrations of liposomal amphotericin B (4 mg/kg) had a longer survival time. Thus, an improved therapeutic index resulted by encapsulating amphotericin B in liposomes.

The National Committee for Clinical Laboratory Standards (NCCLS) proposed macrobroth reference method (M27P) for susceptibility testing of yeasts is technically difficult. We evaluated Etest, a simple agar-based MIC methodology, as a possible alternative. In studies of six yeast quality control strains, Etest yielded results identical to those obtained by the NCCLS reference method for both amphotericin B and fluconazole. In studies of 91 clinical Candida isolates, agreement ؎ 2 dilutions between the two methods was 95% for fluconazole with phosphate-buffered RPMI 1640 agar and 96 to 97% for amphotericin B with either MOPS (morpholinepropanesulfonic acid)-buffered RPMI 1640 agar or antibiotic medium 3 agar. While the two methods had excellent general agreement, testing of a collection of amphotericin B-resistant isolates demonstrated that, unlike the NCCLS reference method, Etest readily identified the resistant isolates and could do so with a defined medium. Etest is equivalent to the NCCLS proposed method for susceptibility testing of yeasts and superior in its ability to detect amphotericin B resistance.In vitro susceptibility testing of yeasts is fraught with numerous problems and affected by factors such as media, buffer, and inoculum density (11). A proposed standard (M27P) using RPMI 1640 medium buffered with morpholinepropanesulfonic acid (MOPS) to pH 7.0 in a macrobroth format with incubation for 48 h at 35ЊC has been published by the National Committee for Clinical Laboratory Standards (NCCLS) (6). This method is time-consuming, expensive, and technically difficult to perform. Modifications of the NCCLS method using a microbroth format will simplify its implementation and have demonstrated results comparable to those for the proposed standard (5). Both of these methods are, however, particularly lacking in their ability to differentiate among amphotericin B-susceptible and -resistant isolates unless antibiotic medium 3 is substituted for RPMI 1640 (9, 11).Etest, a novel method utilizing a stable gradient of an antimicrobial agent, has been well documented as an accurate and simple method for bacterial susceptibility testing (12), and Etest has recently been shown to produce results comparable to those obtained with the NCCLS method for ketoconazole, itraconazole, and fluconazole (3, 4). We have extended this work by comparing the NCCLS method with Etest for testing of susceptibility of Candida isolates to both fluconazole and amphotericin B for a group of clinical isolates as well for a group of recently described quality control strains (7,8). In addition, having recently demonstrated that the NCCLS reference method has a limited ability to detect amphotericin Bresistant isolates unless antibiotic medium 3 is substituted for RPMI 1640 (9), we also studied the ability of Etest to identify these amphotericin B-resistant isolates. MATERIALS AND METHODSIsolates. Study strains included 91 yeast isolates from blood, a subset of those previously described by Rex et al. (9,10). A collection of previously desc...

The performance of the Etest for testing the susceptibilities to caspofungin (MK-0991) of 726 isolates of Candida spp. was assessed against the National Committee for Clinical Laboratory Standards (NCCLS) microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35°C. MICs were determined by Etest for all 726 isolates with RPMI agar containing 2% glucose (RPG) and were read after incubation for 48 h at 35°C. The Candida isolates included Candida albicans (n ‫؍‬ 486), Candida glabrata (n ‫؍‬ 96), Candida tropicalis (n ‫؍‬ 51), Candida parapsilosis (n ‫؍‬ 47), Candida krusei (n ‫؍‬ 11), Candida lusitaniae (n ‫؍‬ 2), and Candida guilliermondii (n ‫؍‬ 33). In addition, a subset of 314 isolates were also tested by Etest using Casitone agar (CAS) and antibiotic medium 3 agar (AM3). The Etest results obtained using RPG correlated well with reference MICs. Overall agreement was 94% with RPG, 82% with CAS, and 79% with AM3. When RPG was used, agreement ranged from 79% for C. parapsilosis to 100% for C. krusei, C. lusitaniae, and C. guilliermondii. When CAS was used, agreement ranged from 0% for C. lusitaniae to 100% for C. glabrata. With AM3, agreement ranged from 0% for C. lusitaniae to 100% for C. guilliermondii. All three media supported growth of each of the Candida species. Etest results were easy to read, with sharp zones of inhibition. In most instances (75%) where a discrepancy was observed between the Etest and the reference method, the Etest MIC was lower. The Etest method using RPG appears to be useful for determining caspofungin susceptibilities of Candida species.

Glycopeptide-intermediate Staphylococcus aureus (GISA) and, in particular, heterogeneous GISA (hGISA) are difficult to detect by standard MIC methods, and thus, an accurate detection method for clinical practice and surveillances is needed. Two prototype Etest strips designed for hGISA/GISA resistance detection (GRD) were evaluated using a worldwide collection of hGISA/GISA strains covering the five major clonal lineages. A total of 150 strains comprising 15 GISA and 60 hGISA strains (defined by population analysis profiles-area under the curve [PAP-AUC]), 70 glycopeptide-susceptible S. aureus (GSSA) strains, and 5 S. aureus ATCC reference strains were tested. For standardized Etest vancomycin (VA) MIC testing, the modified Etest macromethod with VA and teicoplanin (TP) strips tested with a heavier inoculum using brain heart infusion agar (BHI) and two glycopeptide screening agar plates (6 g/ml VA/BHI and 5 g/ml Mueller-Hinton agar [MHA]) were tested in parallel with the two new Etest GRD strips: a VA 32 (0.5-g/ml)-TP 32 (0.5-g/ml) double-sided gradient (E-VA/TP) with one prototype overlaid with a nutrient (E-VA/TP؉S) to enhance the growth of hGISA. The Etest GRD strips were tested with a standard 0.5-McFarland standard inoculum using MHA and MHA plus 5% blood (MHB) and were read at 18 to 24 and 48 h. The interpretive MIC cutoffs used for the new Etest GRD strips at 24 and 48 h were as follows: for GISA, TP or VA, >8, and a standard VA MIC of >6; for hGISA, TP or VA, >8, and a standard VA MIC of <4. The results on MHB at 48 h showed that E-VA/TP؉S had high specificity (94%) and sensitivity (95%) in comparison to PAP-AUC and was able to detect all GISA (n ‫؍‬ 15) and 98% of hGISA (n ‫؍‬ 60) strains. In contrast, the glycopeptide screening plates performed poorly for hGISA. The new Etest GRD strip (E-VA/TP؉S), utilizing standard media and inocula, is a simple and acceptable tool for detection of hGISA/GISA for clinical and epidemiologic purposes.

Mammalian artificial chromosomes (MACs) provide a means to introduce large payloads of genetic information into the cell in an autonomously replicating, non-integrating format. Unique among MACs, the mammalian satellite DNA-based Artificial Chromosome Expression (ACE) can be reproducibly generated de novo in cell lines of different species and readily purified from the host cells' chromosomes. Purified mammalian ACEs can then be re-introduced into a variety of recipient cell lines where they have been stably maintained for extended periods in the absence of selective pressure. In order to extend the utility of ACEs, we have established the ACE System, a versatile and flexible platform for the reliable engineering of ACEs. The ACE System includes a Platform ACE, containing >50 recombination acceptor sites, that can carry single or multiple copies of genes of interest using specially designed targeting vectors (ATV) and a site-specific integrase (ACE Integrase). Using this approach, specific loading of one or two gene targets has been achieved in LMTK(-) and CHO cells. The use of the ACE System for biological engineering of eukaryotic cells, including mammalian cells, with applications in biopharmaceutical production, transgenesis and gene-based cell therapy is discussed.