罕见角膜炎致病真菌的体外药敏试验

目的探索角膜炎罕见致病真菌对于临床常用抗真菌药物的敏感性。方法应用美国临床实验室标准化委员会制订的标准M38-A方案进行体外药敏试验。结果束状刺盘孢对4种抗真菌药的MIC值最低,≤4.0μg/ml;茄病镰刀菌次之;淡紫拟青霉的MIC值最高;茄病镰刀菌和淡紫拟青霉对氟康唑耐药。结论束状刺盘孢对氟康唑、酮康唑、伊曲康唑和两性霉素B体外试验敏感;淡紫拟青霉对酮康唑、伊曲康唑和两性霉素B的MIC值较高;茄病镰刀菌和淡紫拟青霉对氟康唑耐药。     

米卡芬净对分离自中国的念珠菌和曲霉临床株体外抑菌活性的研究

目的了解新型抗真菌药物米卡芬净(micafungin,MFG)对分离自中国的念珠菌和曲霉临床株的体外抑菌活性。方法参照CLSI(Clinical and Laboratory Standards Institute,以前为NCCLS)制定的M27-A2和M38-A方案测定86株念珠菌和35株曲霉的最低抑菌浓度(MIC)或最低有效浓度(MEC)。结果MFG对大多数念珠菌属和曲霉属均有较好的抑菌作用。对念珠菌属的MIC90从高到低依次为:氟康唑(FLC)敏感的白念珠菌、热带念珠菌、光滑念珠菌为0.125μg/ml,FLC耐药和剂量依赖敏感株为0.25μg/ml,克柔念珠菌为0.5μg/ml,近平滑念珠菌8μg/ml,季也蒙念珠菌>16μg/ml。MFG对烟曲霉的MEC90为≤0.03μg/ml,对非烟曲霉的曲霉属MEC90为0.06μg/ml。MFG与唑类药物、两性霉素B(AMB)不存在交叉耐药,对FLC耐药的念珠菌、伊曲康唑耐药的曲霉、AMB不敏感的曲霉均有好的抑菌活性。结论MFG对多数念珠菌属和曲霉属(包括对唑类耐药和AMB不敏感的菌株)有较好的体外抑菌作用。     

In vitro antifungal activities of anidulafungin and micafungin, licensed agents and the investigational triazole posaconazole as determined by NCCLS methods for 12,052 fungal isolates: review of the literature

The echinocandins anidulafungin and micafungin and the triazole posaconazole are currently undergoing phase III clinical trials. Caspofungin and voriconazole have recently been licensed for the treatment of aspergillosis (both agents), other less common mould (voriconazole) and candidal (caspofungin) infections. This review summarizes the published in vitro data obtained by NCCLS or NCCLS modified methods on the in vitro fungistatic and fungicidal activities of these five agents for yeasts and moulds in comparison to the established agents, amphotericin B, fluconazole, itraconazole, and flucytosine. Among the yeasts, the echinocandins have less activity for Candida parapsilosis and Candida guilliermondii, no activity for Cryptococcus neoformans and Trichosporon spp., but good fungistatic and fungicidal activity in vivo and in vitro for most of the other Candida spp.; this fungicidal activity has been reported by minimum fungicidal concentrations (MFCs) or time kill curve results. The new triazoles exhibit good fungistatic activity (but not fungicidal) for most Candida spp., C. neoformans, and Trichosporon spp. For the Aspergillus spp. evaluated, the echinocandins have similar or better fungistatic activity than those of amphotericin B and the triazoles, but fungicidal activity has been demonstrated only with amphotericin B and the triazoles, with the exception of fluconazole. Most studies showed posaconazole and voriconazole minimum inhibitory concentrations (MICs) ranging from 0.25 to 8 microg/ml for non-solani Fusarium spp., while MIC and minimum effective concentration (MEC) endpoints of the echinocandins were >8 microg/ml. The fungistatic activity of the triazoles is also superior to that of the echinocandins for most of the dimorphic fungi and the Zygomycetes. However, micafungin has activity for the mould phase of most dimorphic fungi, but not for the parasitic or yeast phase of Paracoccidioides brasiliensis. The echinocandins appear to have variable and species dependent fungistatic activity for the dematiaceous fungi, but all agents have poor or no activity against most isolates of Scedosporium prolificans. Only amphotericin B exhibit good fungistatic activity against the Zygomycetes. The combination of caspofungin with some triazoles, amphotericin B or liposomal amphotericin B has been synergistic in vitro, in animal models and in patients. Breakpoints are not available for any mould and antifungal agent combination. In vitro/in vivo correlations should aid in the interpretation of these results, but standard testing conditions are needed for the echinocandins, especially for mould testing, to obtain reliable results.     

Itraconazole vs amphotericin B: in vitro comparative evaluation of the minimal inhibitory concentration (MIC) against clinically isolated yeasts

Itraconazole is a triazole compound which, following several clinical trials, has begun to be used for therapy of mycotic infections. This new drug, with a broad-spectrum antifungal activity, can be orally administered. The Authors studied the in vitro susceptibility to amphotericin B and itraconazole of the following clinical isolates of pathogenic yeasts: 100 Candida albicans, 20 C. tropicalis, 20 C. parapsilosis, 8 C. guilliermondii, 6 C. pseudotropicalis, 24 Torulopsis glabrata and 16 Cryptococcus neoformans.Serial two-fold dilutions, from 100 g/ml to 0.04 g/ml, of each drug were prepared in Yeast Nitrogen Base + Glucose 5%, after dissolving the itraconazole in dimethylsulfoxide (DMSO) and amphotericin B in 5% glucose solution. Amphotericin B (MIC₉₀: 3.12 g/ml) was found to have an average in vitro MIC six-fold lower than itraconazole (MIC₉₀: 25 g/ml).Thus, even though itraconazole is active, amphotericin B remains one of the most effective of the antifungal drugs.     

In vitro and in vivo antifungal activities of liposomal amphotericin B,and amphotericin B lipid complex

The in vitro and in vivo antifungal activities of liposomal amphotericin B (L-AMPH) and amphotericin B lipid complex (ABLC), which is composed of amphotericin B and the phospholipids dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol, were compared with those of conventional amphotericin B (Fungizone®, AMPH). The acute intravenous toxicity was markedly lower in BALB/c mice; 50% lethal doses (LD_50s) were 2.75 mg/kg in AMPH, 32.9 mg/kg in L-AMPH and >75 mg/kg in ABLC. In vitro antifungal activities againstCandida albicans, C. parapsilosis, C. tropicalis, C. glabrata, andC. krusei were evaluated by the agar plate dilution method. The activities were unchanged againstC. albicans, but MICs increased more than four fold in 18 of the 20 strains other thanC. albicans in L-AMPH and in 9 of the 20 in ABLC. L-AMPH and ABLC were as efficacious as AMPH in the treatment of mice infected withC. albicans, and at a dose of 0.5 and 1.0 mg/kg of body weight, ABLC was more efficacious on survival. A ten-times larger dose (10 mg/kg) of L-AMPH and ABLC was administered to mice with 100% survival, suggesting improved tolerability as compared to amphotericin B.     

In vitro susceptibilities of clinical yeast isolates to three antifungal agents determined by the microdilution method

A comparative evaluation of the in vitro susceptibilities of 597 clinical yeast isolates to amphotericin B, fluconazole, and 5-fluorocytosine (5FC) was conducted. The broth macrodilution reference method of the National Committee for Clinical Laboratory Standards (NCCLS, M27-P) was adapted to the microdilution method. Microdilution endpoints for amphotericin B were scored as the lowest concentration in which a score of 0 (complete absence of growth) was observed and for 5FC and fluconazole as the lowest concentration in which a score of 2 (prominent decrease in turbidity; MIC-2) was observed compared to the growth control. The MIC values were read after 24 and 48 h incubation. A broad range of MIC values was observed with each antifungal agent. Amphotericin B was very active (MIC₉₀1.0 µg/ml) against all of the yeast isolates with the exception ofC. lusitaniae (MIC₉₀2.0 µg/ml). Fluconazole was most active againstC. parapsilosis (MIC₉₀ of 1.0 µg/ml) and least active againstC. krusei (MIC₉₀ of 32 µg/ml). 5FC was most active againstC. albicans, C. parapsilosis, C. tropicalis, andT. glabrata (MIC₉₀1.0 µg/ml) and was least active againstC. krusei andC. lusitaniae (MIC₉₀16 µg/ml). These data indicate that the microdilution method, performed in accordance with M27-P, provides a means of testing larger numbers of yeast isolates against an array of antifungal agents and allows this to be accomplished in a reproducible and standardized manner. Given these results, it appears that the microdilution method may be a useful alternative to the macrodilution reference method for susceptibility testing of yeasts.     

In vitro assays on the susceptibility of four species of nematophagous fungi to anthelmintics and chemical fungicides/antifungal drug

Using nematophagous fungi for the biological control of animal parasitic nematodes will become one of the most promising strategies in the search for alternative chemical drugs. The purpose of this study was to check the in vitro activity of four anthelmintics, four chemical fungicides and two antifungal drugs on the spore germination of nematophagous fungi: Duddingtonia flagrans (SF170), Arthrobotrys oligospora (447), Arthrobotrys superba (435) and Arthrobotrys sp. (PS011). A modified 24-well cell culture plate assay was conducted to evaluate the susceptibility of nematophagous fungi against drugs tested by calculating the effective middle concentrations (EC₅₀) of each tested drug to inhibit the germination of fungal spores. EC₅₀ ranged between 0·7 and 47·2 μg ml⁻¹ for fenbendazole, thiabendazole and ivermectin, except levamisole (546·5–4057·8 μg ml⁻¹). EC₅₀ of tested fungicides was 0·6–2·3 μg ml⁻¹ for carbendazim, 55·9–247·4 μg ml⁻¹ for metalaxyl, 24·4–45·2 μg ml⁻¹ for difenoconazole, and 555·9–1438·3 μg ml⁻¹ for pentachloronitrobenzene (PCNB). EC₅₀ of two antifungal drugs was 0·03–3·4 μg ml⁻¹ for amphotericin B and 0·3–10·9 μg ml⁻¹ for ketoconazole. The results showed that 10 tested drugs, except for levamisole and PCNB, had in vitro inhibitory effects on nematophagous fungi. The chlamydospores of D. flagrans had the highest sensitivity to nine tested drugs, except for ketoconazole.     

In vitro susceptibility of dermatomycoses agents to six antifungal drugs and evaluation by fractional inhibitory concentration index of combined effects of amorolfine and itraconazole in dermatophytes

To investigate the antifungal drug susceptibility of fungi responsible for dermatomycoses, minimum inhibition concentration (MIC) tests were performed in 44 strains of dermatophytes, including Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton tonsurans, Microsporum canis, Microsporum gypseum and Epidermophyton floccosum, with six antifungal drugs (amorolfine, terbinafine, butenafine, ketoconazole, itraconazole and bifonazole) by broth microdilution assay according to Clinical Laboratory Standard Institute protocols. Six possible dermatomycosis‐causing non‐dermatophytic fungi were also tested. The two major causes of tinea, T. rubrum and T. mentagrophytes, showed significantly different sensitivities to ketoconazole and bifonazole. Clinically derived dermatophytes were sensitive to the six antifungal drugs tested. However, non‐dermatophytes, especially Fusarium spp., tended to be resistant to these antifungal drugs. In Trichophyton spp., the MICs of non‐azole drugs had narrower distributions than those of azoles. To evaluate the effects of antifungal drug combinations, the fractional inhibitory concentration index was calculated for the combination of amorolfine and itraconazole as representative external and internal drugs for dermatophytes. It was found that this combination had synergistic or additive effects on most dermatophytes, and had no antagonistic effects. The variation in susceptibility of clinically derived fungal isolates indicates that identification of causative fungi is indispensable for appropriately choosing effective antifungal drugs in the early stages of infection. The results of combination assay suggest that multiple drugs with different antifungal mechanisms against growth of dermatophytes should be used to treat refractory dermatomycoses, especially onychomycosis.     

In vitro activity of the lipopeptide PAL-Lys-Lys-NH2, alone and in combination with antifungal agents, against clinical isolates of Candida spp

Candida albicans is known to be the organism most often associated with serious fungal infection, but other Candida spp. are emerging as clinical pathogens associated with opportunistic infections. Among antimycotic treatments, increasing attention is currently given to anti-infective drugs based upon naturally occurring peptides, such as the short lipopeptide palmitoyl PAL-Lys-Lys-NH2 (PAL). The aim of this study is to evaluate the activity of this peptide compared to the traditional antifungal agents Fluconazole (FLU), amphotericin B (AMB) and caspofungin (CAS) on Candida spp. 24 clinical isolates of Candida spp. were tested against PAL_, FLU, AMB and CAS using in vitro susceptibility tests, time killing and checkerboard assay. All of the drugs studied showed good activity against clinical isolates of candida; in particular CAS and AMB which have MICs value lower than PAL and FLU. Moreover we observed synergistic interactions for PAL/FLU (81.25%), PAL/AMB (75%) and particularly for PAL/CAS (87.5). We think that our results are interesting since synergy between PAL and CAS might be useful in clinic trails to treat invasive fungal infections.     

Reversible binding interactions between the tryptophan enantiomers and albumins of different animal species as determined by novel high performance liquid chromatographic methods: an attempt to localize the D- and L-tryptophan binding sites on the human serum albumin polypeptide chain by using protein fragments

The stereoselectivity of the reversible binding interactions between the D- and L-tryptophan enantiomers and serum albumins of different animal species and fragments of human serum albumin (HSA) was investigated by applying three novel high performance liquid chromatographic (HPLC) arrangements. The separations were performed by means of (1) an achiral (diol-bond), (2) a chiral (bovine serum albumin-bond) silica gel sorbent, and (3) a column switching technique which uses both the diol- and HSA-bond HPLC stationary phases. A polarimetric detector and/or an ultraviolet (UV) spectrophotometer were used to monitor the separation process. HPLC arrangement 3 allowed the evaluation of enantioselective binding for D- and L-tryptophan to different albumins and albumin fragments. At present, column switching can be considered the technique of the broadest applicability for investigating the reversible binding interactions between a protein and drug enantiomers. Chirality 9:373–379, 1997. © 1997 Wiley-Liss, Inc.