Direct comparison of the pharmacodynamics of four antifungal drugs in a mouse model of disseminated candidiasis using microbiological assays of serum drug concentrations

The aim of this study was to compare the pharmacodynamics of the azole antifungal drugs fluconazole, itraconazole and ketoconazole, and the polyene antifungal amphotericin B, in a mouse model of disseminated Candida albicans infection. In order to directly compare effective serum concentrations of these antifungals, drug concentrations were assayed microbiologically by measuring inhibition of C. albicans mycelial growth (mMIC) in a mouse serum-based assay (serum antifungal titer). Efficacy in the mouse infection model was determined using an organ-based (kidney burden) endpoint. For all four drugs, the serum antifungal titers, 8 hr after administration of single doses of drugs at a range of drug concentrations, correlated closely with C. albicans kidney fungal burden in the mouse model. The results showed that determining serum antifungal titer may be used to accurately represent kidney fungal burden in a mouse model of disseminated candidiasis and allowed direct comparison of the pharmacodynamics of differing classes of antifungal drugs.     

地塞米松影响念珠菌对抗真菌药物敏感性的实验研究

目的 探讨地塞米松在体外试验中是否影响念珠菌对抗真菌药物的敏感性,以了解糖皮质激素与抗真菌药物直接作用于念珠菌时是否存在相互作用。方法 用微量液体培养基稀释法分别测定26株白念珠菌与地塞米松（0．2mg／ml）共同孵育前、孵育24～48h及7d时氟康唑、伊曲康唑、两性霉素B的最低抑菌浓度（MIC）值,并作对照。结果 白念珠菌与地塞米松孵育24～48h后、孵育后第7d氟康唑和伊曲康唑的MIC值升高,分别与孵育前的MIC值存在统计学差异,但孵育24～48h后的MIC与孵育后第7d的MIC无统计学差异;白念珠菌与地塞米松共同孵育24～48h后两性霉素B的MIC值也较孵育前升高,但第7d的MIC值与孵育前无差异。结论 地塞米松可增加三种抗真菌药物对于白念珠菌的MIC,但三种抗真菌药物间存在差异,表明地塞米松对于氟康唑和伊曲康唑体外抗白念珠菌的活性有拮抗作用,但没有时间依赖性,地塞米松对于两性霉素B的影响较氟康唑和伊曲康唑小,且影响时间较短。     

Evaluation of antifungal pharmacodynamic characteristics of AmBisome against Candida albicans

A liposomal formulation of Amphotericin B (AmBisome), with small unilamellar vesicles containing amphotericin B, shows characteristic pharmacokinetics as liposomes, and in consequence, has different pharmacological activity and toxicity from amphotericin B deoxycholate (Fungizone). In this study, we evaluated the antifungal pharmacodynamic characteristics of AmBisome against Candida albicans using the in vitro time-kill method and murine systemic infection model. A time-kill study indicated that the in vitro fungicidal activities of AmBisome and Fungizone against C. albicans ATCC 90029 increased with increasing drug concentration. For in vivo experiments, leucopenic mice were infected intravenously with the isolate 4 hr prior to the start of therapy. The infected mice were treated for 24 hr with twelve dosing regimens of AmBisome administered at 8-, 12-, 24-hr dosing intervals. Correlation analysis between the fungal burden in the kidney after 24 hr of therapy and each pharmacokinetic/pharmacodynamic parameter showed that the peak level/MIC ratio was the best predictive parameter of the in vivo outcome of AmBisome. These results suggest that AmBisome, as well as Fungizone, has concentration-dependent antifungal activity. Furthermore, since AmBisome can safely achieve higher concentrations in serum than Fungizone, AmBisome is thought to have superior potency to Fungizone against fungal infections.     

Multicenter survey of in vitro antifungal resistance in yeasts of medical importance isolated from Spanish patients

Twelve Spanish laboratories collected 325 yeast clinical isolates during a 30 day's period, among them 224 Candida albicans, 30 Candida glabrata, and 27 Candida parapsilosis. In vitro antifungal susceptibility to amphotericin B, ketoconazole, fluconazole and itraconazole was determined by an agar diffusion test (Neo-Sensitabs, Rosco, Denmark). All the isolates tested were susceptible in vitroto amphotericin B and nearly all (97.2%) to itraconazole. In vitrosusceptibility to fluconazole and ketoconazole was high (90.2% and 91.4% of isolates, respectively) but showed variations depending on the species tested. Resistance to fluconazole and ketoconazole was low in C. albicans (4% and 3%, respectively), but 30% of Candida guilliermondii and 36% of C. glabrata isolates were resistant to fluconazole. Ketoconazole resistance was observed in 40% of C. glabrata, and 17% of Candida tropicalis. Resistance to antifungal drugs is very low in Spain and it is related to non-C. albicans isolates.     

Effect of subinhibitory concentration of some established and experimental antifungal compounds on the germ tube formation in<Emphasis Type="Italic">Candida albicans</Emphasis>

The influence of subinhibitory concentrations of six established and 19 newly synthesized antifungal compounds on the dimorphic transition of three C. albicans strains was evaluated in the filamentation-inducing medium. Amphotericin B was found to produce almost complete inhibition in the germination at a concentration of 1/10 of the corresponding MIC and partial inhibition at a concentration as low as MIC/50. Flucytosine and four azole derivatives were proven ineffective. From the newly synthesized drugs, the incrustoporin derivative LNO6-22, two phenylguanidine derivatives (PG15, PG45), and four thiosalicylanilide derivatives, in particular, showed results comparable to those of amphotericin B, with a high inhibition of germ tube formation at concentrations of MIC/10. In general, concentrations of MIC/50 had no visible effect.     

额外拷贝ERG6基因对烟曲霉的影响

通过构建烟曲霉ERG6基因额外拷贝株．研究该基因对烟曲霉生长速度、抗药物敏感性的影响。在烟曲霉基因组找出烟曲霉可能的ERG6基因的开放读码框(ORF),PCR扩增ERG6的ORF连同其上下游各约1 kb的DNA片段,利用DNA重组的方法将该片段克隆到载体pRG-AMA1-NotI。用重组后的质粒转化烟曲霉尿嘧啶营养缺陷株AF293．1。在MM和YAG培养基上观察转化子的生长速度。采用纸片扩散法和微量液基稀释法测定转化子对抗真菌药物敏感性。烟曲霉基因组中存在一个拷贝的ERG6基因,ORF大小为1,256 bp。其编码的蛋白与白念珠菌、酿酒酵母固醇甲基转移酶(Ers6p)的氨基酸相同率分别为57%和50%,相似率分别为70%和63%。烟曲霉中ERG6基因被成功克隆到了pRG-AMA1-Not I,产生了质粒pERG6。用pERG6和空载体pRG-AMA1-Not I转化AF293．1后,分别得到转化子AF-pERG6和AF-empty。AF-pERG6在MM和YAG培养基上的生长速度均比AF-empty慢。AF-pERG6和AF-empty对伊曲康唑、伏力康唑、特比萘芬、两性霉素B、卡泊芬净、灰黄霉素的敏感性没有差异。ERG6基因额外拷贝不影响烟曲霉对伊曲康唑、伏力康唑、特比萘芬、两性霉素B、卡泊芬净、灰黄霉素的敏感性,但是能使烟曲霉的生长速度减慢。     

小鼠烟曲霉角膜炎的实验研究

目的 比较伊曲康唑和氟康唑对烟曲霉的体外抗菌活性,观察伊曲康唑对小鼠烟曲霉角膜炎的治疗作用.方法 通过角膜基质注射法建立烟曲霉角膜炎小鼠模型.造模后观察角膜病变,取角膜病变处分泌物做真菌镜检、真菌培养以证实造模成功.用药基法检测伊曲康唑和氟康唑对烟曲霉的最低抑菌浓度( MIC)和最低杀菌浓度(MFC).对烟曲霉角膜炎小鼠给予伊曲康唑治疗,治疗结束行临床评分、炎性评分、菌落形成单位测定以评价疗效.结果 伊曲康唑对烟曲霉的MIC和MFC分别为6.25 μg/mL、12.5 μg/mL;氟康唑对烟曲霉的MIC和MFC分别为500 μg/mL、1 000 μg/mL.伊曲康唑治疗组临床评分、炎性评分和测定的菌落数较对照组均明显减少(P＜0.05).结论 伊曲康唑对烟曲霉的体外抗菌活性优于氟康唑,并且对烟曲霉性角膜炎有明显疗效.     

Use of spectrophotometric reading for in vitro antifungal susceptibility testing of Aspergillus spp

A comparative study of visual and spectrophotometric MIC endpoint determinations for antifungal susceptibility testing of Aspergillus species was performed. A broth microdilution method adapted from the National Committee for Clinical Laboratory Standards (NCCLS) was used for susceptibility testing of 180 clinical isolates of Aspergillus species against amphotericin B and itraconazole. MICs were determined visually and spectrophotometrically at 490 nm after 24, 48, and 72 h of incubation, and MIC pairs were compared. The agreement between the two methods was 99% for amphotericin B and ranged from 95 to 98% for itraconazole. It is concluded that spectrophotometric MIC endpoint determination is a valuable alternative to the visual reference method for susceptibility testing of Aspergillus species.     

Use of a serum-based antifungal susceptibility assay to predict the in vivo efficacy of novel echinocandin compounds

In vitro susceptibility assays of antifungal activity do not always accurately predict in vivo efficacy. As well as having a clear clinical importance, the ability to predict efficacy is also essential for effective screening of novel drug compounds. Initial screening of novel compounds must often be based on in vitro data. The present report describes the use of serum-MIC, an in vitro test of antifungal susceptibility, to accurately predict in vivo efficacy of echinocandin drugs in a mouse model of disseminated candidiasis. The basis of the serum-MIC method was to measure the inhibitory activity of a test compound against Candida albicans hyphal growth in the presence of pooled mouse serum. For 13 previously uncharacterized echinocandin compounds, as well as for the known echinocandin drugs, micafungin and caspofungin, serum-MIC determinations were shown to give better correlation to efficacy in the animal model than conventional, CLSI standard, in vitro antifungal susceptibility tests. The most accurate prediction of efficacy was obtained when the serum-MIC was adjusted in relation to the serum concentration at 30 min post-treatment. Furthermore, when the efficacy of micafungin was determined by measuring C. albicans kidney burden in the mouse model of infection, the adjusted serum-MIC consistently reflected the effective serum concentrations. Our data indicate that determination of serum-MIC values will facilitate prediction of the in vivo potency of new antifungal compounds such as novel echinocandins.     

In vitro activities of voriconazole, fluconazole, itraconazole and amphotericin B against non Candida albicans yeast isolates

Antifungal susceptibility testing was performed on 197 yeast isolates from the BCCM/IHEM biomedical fungi and yeasts collection (Belgian Co-ordinated Collections of Micro-organisms / IPH-Mycology) to study the in vitro activity of voriconazole against fluconazole, itraconazole and amphotericin B. MICs of the four antifungal agents were determined by an adapted NCCLS M27-A microdilution reference method. MIC readings were visually and spectrophotometrically determined. Optical density data were used for calculation of the MIC endpoints. For amphotericin B, the MIC endpoint was defined as the minimal antifungal concentration that exerts 90% inhibition, compared to the control growth. The azoles endpoints were determined at 50% inhibition of growth. The MIC distribution of voriconazole susceptibilities showed that 193 isolates had a MIC < or = 2 microg/ml and 185 a MIC < or = 1 microg/ml. Cross-tabulation of voriconazole, fluconazole, and itraconazole MICs indicated that voriconazole MICs raised with fluconazole and itraconazole MICs. The in vitro data obtained in this study suggest that voriconazole may also be effective treating yeast infection in patients infected with fluconazole or itraconazole resistant isolates.     

ATB FUNGUS2法在念珠菌属和新生隐球菌抗真菌药敏试验中应用的评价

目的评价ATBFUNGUS2半固体培养基法在测定念珠菌属和新生隐球菌对4种常用抗真菌药物敏感性中的应用价值。方法利用CLSIM27．A2微量液基稀释法和ATBFUNGUS2法同时测定131株念珠菌和20株新生隐球菌对两性霉素B（AmB）、氟康唑（FLC）、氟胞嘧啶（5-Fc）和伊曲康唑（ITC）的敏感性。结果①两种方法对于AmB、5-FC、FLC和ITC的一致性分别为98％、89．4％、78．8％和78．1％;②所有受试菌株中两种方法的一致性为80％,但ATBFUNGUS2法将2／5株M27-A2法检查为FLC耐药的白念珠菌判断为敏感或剂量依赖,将8／10株M27-A2法检查为FLC剂量依赖的白念珠菌判断为敏感或耐药。③ATBFUNGUS2法中AmB的MIC值判读范围偏高,以致于实际工作中不能读出准确的值。结论ATBFUNGUS2半固体培养基法在测定念珠菌属和新生隐球菌对4种常用抗真菌药物的敏感性时不失为简单、快速而且重复性好的方法。     

Development of Reference Procedures for Broth Microdilution Antifungal Susceptibility Testing of Yeasts with Standardized Endpoint Determination

Standard guidelines for the broth microdilution antifungal susceptibility testing of amphotericin B, flucytosine, fluconazole, miconazole and itraconazole are reported. These are a modification of the method developed by the National Committee for Clinical Laboratory Standards (NCCLS) on the following two points: standardization of the means of endpoint determination and the inclusion of miconazole and itraconazole in the testing. MIC was determined to be when the positive control had a turbidity of 0.2 at the 630 nm wavelength. The endpoint was 80% inhibition for azoles and 100% inhibition for other drugs. The method provided good reproducibility, and a wide range of MIC distribution was observed in all antifungal agents except amphotericin B.     

In vitro antifungal susceptibility of clinical isolates of Candida spp. obtained from patients with different predisposing factors to candidosis

The increase in the number of infections caused by Candida species and the consequent use of antifungal agents favours an increase of resistant isolates. The aim of this study was to evaluate the antifungal susceptibility of Candida spp. isolates from patients with different systemic predisposing factors to candidosis. Seventy-nine Candida spp. isolates were assayed for in vitro susceptibility to amphotericin B, fluconazole, 5-flucytosine and itraconazole using the technique proposed by the Clinical and Laboratory Standards Institute (CLSI). Four C. albicans, one C. guilliermondii, four C. parapsilosis and two C. tropicalis isolates were resistant to amphotericin B. Only two isolate was resistant to itraconazole. All the isolates tested were susceptible to fluconazole and flucytosine. It could be concluded that the most efficient drugs against the Candida isolates studied were fluconazole and flucytosine and that all of the antifungal agents used in this study were effective against the Candida spp. isolates tested.     

In vitro activity of a new triazole antifungal agent,Sch 56592, against clinical isolates of filamentous fungi

Sch 56592 is a new triazole derivative that possesses potent, broad-spectrum antifungal activity. We evaluated the in vitroactivity of Sch 56592 compared with that of itraconazole, amphotericin B and 5-fluorocytosine against 51 clinical isolates of filamentous fungi, including Aspergillus flavus(10), A. fumigatus(12), Fusariumspp. (13), Rhizopus spp. (6), Pseudallescheria boydii(5), and one isolate each of Acremoniumspp., A. niger, A. terreus, Paecilomycesspp., and Trichodermaspp. In vitrosusceptibility testing was performed using the microdilution broth method outlined in the NCCLS 27-A document. Sch 56592 was highly active against A. flavus(MIC₉₀, 0.25 μg/ml), A. fumigatus(MIC₉₀, 0.12 μg/ml), P. boydii(MIC₅₀, 1 μ/ml) and Rhizopusspp (M1C₅₀, 1 μg/ml). By comparison with itraconazole, Sch 56592 was four- to eight-fold more active against isolates of Aspergillusand both compounds showed equipotent in vitroactivity against P. boydiiand Rhizopusspp. Sch 56592 was four- to 16-fold more active than amphotericin B against Aspergillusspp. and P. boydiiand both antifungal drugs displayed similar activity against Rhizopusspp. Overall, Sch 56592 showed good in vitroactivity against all isolates tested (MIC, ≤ 2 μg/ml) except isolates of Fusarium(MIC range, 1–>4 μg/ml). On the basis of these data Sch 56592 has promising activity against Aspergillus spp. and other species of filamentous fungi that are likely to be encountered clinically. Additional in vitroand in vivostudies are warranted. This revised version was published online in June 2006 with corrections to the Cover Date.     

新疆地区白念珠菌基因型分析及其体外药物敏感性研究

目的研究新疆地区汉族和维吾尔族患者来源的50株白念珠菌的基因型及其对两性霉素B、5-氟胞嘧啶、米卡芬净、伊曲康唑、氟康唑和咪康唑的体外敏感性。方法采用PCR法扩增白念珠菌rDNA 25S的Ⅰ类内含子包含区,根据扩增产物的大小判断基因型(A型为450 bp,B型为840 bp,C型为450 bp和840 bp)。采用CLSI M27-A液基微量稀释法测定50株白念珠菌对上述6种抗真菌药的体外敏感性。结果 50株菌分为3种基因型:A型30株,B型和C型各10株。所有菌株对两性霉素B、5-氟胞嘧啶、米卡芬净和咪康唑的MIC值较低,MIC范围依次为0.25～0.5μg/mL,0.125～0.5μg/mL,≤0.03μg/mL,0.25～8μg/mL;对伊曲康唑和氟康唑的MIC值较高,MIC范围分别为0.25～8μg/mL,0.5～64μg/mL。B型和C型对5-氟胞嘧啶的MIC值均为0.125μg/mL,对伊曲康唑和氟康唑的耐药率分别为84%、70%。不同族别来源的菌株基因型比较无显著差异(P>0.05),不同基因型菌株的抗真菌药物敏感性比较也无显著差异(P>0.05)。结论新疆地区白念珠菌分A,B,C三种基因型。汉...     

Synthesis and investigation of novel benzimidazole derivatives as antifungal agents

The rise and emergence of resistance to antifungal drugs by diverse pathogenic fungal strains have resulted in an increase in demand for new antifungal agents. Various heterocyclic scaffolds with different mechanisms of action against fungi have been investigated in the past. Herein, we report the synthesis and antifungal activities of 18 alkylated mono-, bis-, and trisbenzimidazole derivatives, their toxicities against mammalian cells, as well as their ability to induce reactive oxygen species (ROS) in yeast cells. Many of our bisbenzimidazole compounds exhibited moderate to excellent antifungal activities against all tested fungal strains, with MIC values ranging from 15.6 to 0.975 μg/mL. The fungal activity profiles of our bisbenzimidazoles were found to be dependent on alkyl chain length. Our most potent compounds were found to display equal or superior antifungal activity when compared to the currently used agents amphotericin B, fluconazole, itraconazole, posaconazole, and voriconazole against many of the strains tested.     

男性尿道炎和包皮龟头炎致病真菌的分布与药敏分析

目的了解男性念珠菌性尿道炎和包皮龟头炎的菌群分布及体外抗真菌药敏试验情况。方法菌株分离均来自复旦大学附属华山医院皮肤性病门诊临床症状轻重不一、真菌直接镜检阳性的61例患者。用科玛嘉念珠菌显色培养基及API 20C AUX鉴定系统进行菌种鉴定;采用CLSIM27-A2肉汤微量稀释法对61株临床分离念珠菌作了氟康唑、两性霉素B、氟胞嘧啶、伊曲康唑、伏立康唑、特比萘芬6种抗真菌药物敏感性测定。结果对培养阳性的61例菌株,通过科玛嘉念珠菌显色培养基及API 20C AUX鉴定系统作菌种鉴定,白念珠菌52例（85.2%）,近平滑念珠菌3例,光滑念珠菌2例,热带念珠菌2例,季也蒙念珠菌1例,克柔念珠菌1例。对52株白念珠菌的药敏试验显示氟康唑98.1%敏感,1.9%剂量依赖性敏感;氟胞嘧啶96.2%敏感,3.8%耐药;伊曲康唑44.2%敏感,40.5%剂量依赖性敏感,15.3%耐药;伏立康唑84.6%敏感,15.4%耐药;两性霉素B全部敏感;特比萘芬的MIC范围为1-64μg/ml,MIC50和MIC90皆为64μg/ml。结论在男性念珠菌性尿道炎和包皮龟头炎中,白念珠菌仍是第一位致病菌,体外药敏试验显示氟康唑、伏立康唑、氟胞嘧啶、两性霉素B对男性念珠菌性尿道炎均有较好的敏感性。     

Synthesis and antifungal activity of substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles

Series of substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles derivatives have been synthesized and examined for their activity against pathogenic strains of Aspergillus fumigatus (ITCC 4517), Aspergillus flavus (ITCC 5192) Aspergillus niger (ITCC 5405) and Candida albicans (ITCC No 4718). All synthesized compounds showed mild to moderate activity, except for 2-substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles 6a-d. The most active 1-(4-chlorophenyl)-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indole 4c exhibited a MIC value of 5.85 microg/disc against A. fumigatus and 11.71 microg/disc against A. flavus and A. niger in disc diffusion assay. Anti-Aspergillus activity of active compound 4c by microbroth dilution assay was found to be 15.62 microg/ml in case of A. fumigatus and 31.25 microg/ml with A. flavus and A. niger. The MIC90 value of the most active compound by percent germination inhibition assay was found to be 15.62 microg/ml against A. fumigatus. The MIC90 values of substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles against C. albicans ranged from 15.62 to 250 microg/ml. The in vitro toxicity of the most active 1-(4-chlorophenyl)-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indole 4c was evaluated using haemolytic assay, in which the compound was found to be non-toxic to human erythrocytes up to a concentration of 312.50 microg/ml. The standard drug amphotericin B exhibited 100% lysis at a concentration of 37.5 microg/ml.     

Streptomyces halstedii K122 produces the antifungal compounds bafilomycin B1 and C1

Streptomyces halstedii K122 was previously found to produce antifungal compounds on solid substrates that inhibit radial growth of fungi among Ascomycetes, Basidiomycetes, Deuteromycetes, Oomycetes, and Zygomycetes, and strongly affected hyphal branching and morphology. During growth of S. halstedii K122 in submerged culture, no antifungal activity could be detected. However, cultivation of S. halstedii in thin (1 mm) liquid substrate layers in large surface-area tissue culture flasks caused intense growth and sporulation of S. halstedii K122, and the biologically active compounds could be extracted from the mycelium with methanol. Antifungal compounds were purified using C18 solid phase extraction and silica gel column chromatography, and identified as bafilomycins B1 and C1, using 2D NMR and FAB MS. Production of bafilomycins, which are specific inhibitors of vacuolar ATPases, has not been reported from S. halstedii previously. Minimum inhibitory concentrations (MIC) of bafilomycins B1 and C1, amphotericin B, and nikkomycin Z were determined at pH 5.5 and 7.0 for the target fungi Aspergillus fumigatus, Mucor hiemalis, Penicillium roqueforti, and Paecilomyces variotii. Penicillium roqueforti was the most sensitive species to all the compounds investigated. The MIC values for amphotericin B were 0.5-4 micrograms.mL-1 for the fungi tested, and pH did not affect the toxicity. The MIC values for nikkomycin Z ranged from < 0.5 microgram.mL-1 for Mucor hiemalis to > 500 micrograms.mL-1 for Aspergillus fumigatus, and pH had no influence on toxicity. Bafilomycins B1 and C1 were equally active against the fungal species tested, with MIC values in the range of < 0.5-64 micrograms.mL-1. All fungi were more sensitive to both bafilomycin B1 and C1 at pH 7.0 than at pH 5.5.     

Influence of antifungal pre-treatment in preparing test mycelium on MIC values of several antifungal agents againstAspergillus niger

Antifungal activity of two imidazoles (miconazole and ketoconazole) and one polyene (amphotericin B) was evaluated using an automatic growth analysis system. Spores ofAspergillus niger were inoculated on the polylysine-coated glass bottom of a culture vessel. A colony formed in liquid medium was exposed to an antifungal agent and subsequently washed. Based on the dynamic growth rate of a test hypha selected from the colony in response to the antifungal agent, the minimum inhibitory concentration (MIC) was evaluated. The influence of time of reading (1, 2 and 3 h after washing) on the MIC determined was investigated. MICs for test hyphae subjected to antifungal pre-treatment were compared with those for hyphae without pre-treatment. Hyphae pre-treated with an antifungal agent for 1 h were found to become adapted and tolerant to that antifungal agent. Hyphae exposed and adapted to an imidazole obtained tolerance to amphotericin B as well as to the other imidazole.