棘白菌素对氟康唑耐药的念珠菌体外药物敏感性的研究

目的评价3种棘白菌素类药物（卡泊芬净、米卡芬净、阿尼多芬净）体外对氟康唑耐药念珠菌的药物敏感性。方法采用微量液体稀释法和琼脂稀释法测定最小抑制浓度（MIC）。结果微量液体稀释法：59株耐药白念珠菌3种药物MIC50均为0.06μg/mL,米卡芬净、阿尼多芬净的MIC范围均为0.015～0.125μg/mL,卡泊芬净为0.015～0.25μg/mL;8株耐药光滑念珠菌MIC值均为0.063μg/mL。琼脂稀释法：59株耐药白念珠菌和8株耐药光滑念珠菌3种药物MIC值均为0.063μg/mL。结论3种棘白菌素类药物可能具有治疗氟康唑耐药的念珠菌感染的临床价值。     

卡泊芬净、米卡芬净对念珠菌体外药物敏感性的动态研究

目的 动态研究卡泊芬净、米卡芬净体外对念珠菌的药物敏感性.方法 参照CLSI公布的M-27A方案微量液体稀释法分别测定卡泊芬净、米卡芬净、氟康唑对85株念珠菌的体外敏感性,并连续7d观测结果.结果 48 h卡泊芬净对白念珠菌、光滑念珠菌及其他念珠菌MIC50、MIC90中位数分别为0.030μg/mL、0.030 μg/mL,0.060μg/mL、0.125 μg/mL,0.125 μg/mL、0.500 μg/mL.48 h米卡芬净对白念珠菌、光滑念珠菌及其他念珠菌MIC50、MIC90中位数分别为0.030 μg/mL、0.030 μg/mL,0.060 μg/mL、0.060 μg/mL,0.250 μg/mL、0.500 μg/mL.48 h氟康唑对白念珠菌、光滑念珠菌及其他念珠菌MIC80、MIC100中位数分别为2μg/mL、128 μg/mL,64 μg/mL、128 μg/mL,2μg/mL、32μg／mL.85株念珠菌中未见对3种药物同时耐药的菌株.卡泊芬净组白念珠菌MIC50、MIC90 24 h后不再升高;光滑念珠菌MIC50 72 h后不再升高,MIC90 120 h后不再升高;其他念珠菌组MIC50 168 h、MIC90 96 h后不再升高.米卡芬净组白念珠菌、光滑念珠菌MIC50、MIC90 24 h后不再升高;其他念珠菌MIC50、MIC90在72 h后不再升高.结论卡泊芬净、米卡芬净对念珠菌属有较好的抗菌作用,其中对白念珠菌、光滑念珠菌作用更强,且MICs随着作用时间延长而升高并存在药物特异性和念珠菌种属特异性.     

白念珠菌SSK1突变株对氟康唑敏感性的初步研究

目的探讨氟康唑作用于白念珠菌双组分信号传导途径SSK1突变株SSK21后药物敏感性的变化。方法采用微量液体稀释法和固醇测定法测定野生株(CAF2-1)和突变株(SSK21)的最小抑菌浓度(MIC);并应用RT-PCR观察氟康唑作用前后,SSK1的表达变化。结果氟康唑对CAF2-1的MIC为16μg/mL,对SSK21为0.032μg/mL。加入氟康唑后,CAF2-1的SSK1表达明显增加,60min时达到最多。结论 SSK21对氟康唑高度敏感,SSK1基因及其相关的重要基因与药物敏感性的关系值得进一步研究,从而为新的抗真菌药物和治疗途径的研发提供参考。     

Minimal inhibitory concentrations of Candida species to five antifungals by using the reference dilution micromethod and the E-test.

It is accepted that the frequency of candidosis has increased during the last decade, specially in hospitalized patients. The more frequent use of azole antifungals and the recognition of isolates of Candida sp resistant to these and other drugs such as 5-fluorocytosine constitute a great need for a reproducible and useful C. albicans in vitro susceptibility testing method for monitoring antifungal therapy in clinical mycological laboratories. The E-test is a novel agar diffussion technique for testing the susceptibility of yeasts against a defined continous gradient of drug and could be used by most clinical laboratories. In this study the E-test and the NCCLS reference microbroth method (M27-P guidelines) were used to determine the MICs of amphotericin B, 5-flucytosine, itraconazole, fluconazole and ketoconazole for 50 clinical isolates of Candida albicans, Torulopsis glabrata, C. tropicalis and Hansenula anomala and five reference ATCC strains. The main purpose of the study was to compare the results obtained by the two methods. In general good agreement (+/- 1 dilution) was otained between both methods, despite differences observed for some species-antifungal combinations in which the MICs were lower by the E-test than by the microbroth method. MICs for C. albicans and T. glabrata to amphotericin B were < 0.50 microg/mL. Two isolates of C. albicans and two others of H. anomala, showed MIC < 8 microg/mL for 5- flucytosine. All isolates of T. glabrata and 40% of C. albicans showed MICs > 16 microg/mL for fluconazole. The results of this study indicate that E-test is an alternative for susceptibility testing to the NCCLS reference method. Because its simplicity it seems to be an easier test for routine clinical laboratories.     

Carboxylic acid and phosphate ester derivatives of fluconazole: synthesis and antifungal activities

Two classes of fluconazole derivatives, (a) carboxylic acid esters and (b) fatty alcohol and carbohydrate phosphate esters, were synthesized and evaluated in vitro against Cryptococcus neoformans, Candida albicans, and Aspergillus niger. All carboxylic acid ester derivatives of fluconazole (1a-l), such as O-2-bromooctanoylfluconazole (1g, MIC=111 microg/mL) and O-11-bromoundecanoylfluconazole (1j, MIC=198 microg/mL), exhibited higher antifungal activity than fluconazole (MIC > or = 4444 microg/mL) against C. albicans ATCC 14053 in SDB medium. Several fatty alcohol phosphate triester derivatives of fluconazole, such as 2a, 2b, 2f, 2g, and 2h, exhibited enhanced antifungal activities against C. albicans and/or A. niger compared to fluconazole in SDB medium. For example, 2-cyanoethyl-omega-undecylenyl fluconazole phosphate (2b) with MIC value of 122 microg/mL had at least 36 times greater antifungal activity than fluconazole against C. albicans in SDB medium. Methyl-undecanyl fluconazole phosphate (2f) with a MIC value of 190 microg/mL was at least 3-fold more potent than fluconazole against A. niger ATCC 16404. All compounds had higher estimated lipophilicity and dermal permeability than those for fluconazole. These results demonstrate the potential of these antifungal agents for further development as sustained-release topical antifungal chemotherapeutic agents.     

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.     

特比萘芬与其他抗真菌药物对外阴阴道念珠菌病病原菌的体外相互作用研究

目的 了解特比萘芬( TEB)分别与氟康唑(FLU)、伊曲康唑(ITR)、咪康唑(MCZ)、克霉唑(CMZ)及制霉菌素(NYS)联合对外阴阴道念珠菌病患者分离的念珠菌体外相互作用.方法 受试念珠菌株46株,包括白念珠菌(20株)、热带念珠菌(15株)、光滑念珠菌(4株)、克柔念珠菌(3株)、近平滑念珠菌(3株)和季也蒙念珠菌(1株).方法参考CLSIM27-A3方案中的微量液基稀释法,观察各药单独和联合后的MIC,并计算FICI.结果 TEB、FLU、ITR、MCZ、CMZ和NYS 的单药MIC范围分别为0.5～256μg/mL、0.5～ 128 μg/mL、0.5 ～ 32.0 μg/mL,0.125 ～ 16μg/mL、0.03 ～ 4.0 μg/mL和2～8μg/mL.当TEB与FLU、ITR、MCZ和CMZ联合时,对白念株菌表现为协同作用的分别为100％ (20/20),80％ (16/20),55％ (11/20)和90％ (18/20);对1株季也蒙念珠菌均显示为协同作用;对热带念珠菌,TEB分别与上述4种唑类药物联合时,显示有协同作用的分别为67％ (10/15),60％ (9/15),20％(3/15)和20％ (3/15);TEB与唑类药物联合对光滑念珠菌、克柔念珠菌和近平滑念珠菌均无协同作用;TEB与NYS联合时对所有菌株均无协同作用.结论 TEB与唑类药物联合对不同种的念珠菌显示有不同的效应,但对外阴阴道念珠菌病主要的致病菌白念珠菌显示有良好的协同作用.TEB与NYS联合对念株菌均无协同作用.     

Carbon analogs of antifungal dioxane-triazole derivatives: synthesis and in vitro activities

A new series of triazole compounds possessing a carbon atom in place of a sulfur atom were efficiently synthesized and their in vitro antifungal activities were investigated. The carbon analogs showed excellent in vitro activity against Candida, Cryptococcus, and Aspergillus species. The MICs of compound 1c against C. albicans ATCC24433, C. neoformans TIMM1855, and A. fumigatus ATCC26430 were 0.016, 0.016, and 0.125 microg/mL, respectively (MICs of fluconazole: 0.5, >4, and >4 microg/mL; MICs of itraconazole: 0.125, 0.25, and 0.25 microg/mL).     

Curcumin as a promising anticandidal of clinical interest

Curcumin, an important Asian spice, is part of many Indian food preparations. This work evaluates the antifungal activity of curcumin against 14 strains of Candida (10 clinical and 4 standard). Curcumin displayed antifungal properties against all tested Candida strains, with minimum inhibitory concentrations (MICs) varying from 250 to 2000 μg·mL?1. The in vitro effect of curcumin on growth, sterol content, proteinase secretion, and H+ extrusion by plasma membrane ATPase was investigated for 2 standard strains Candida albicans ATCC 10261 and Candida glabrata ATCC 90030 and compared with the effect of fluconazole. At MIC, curcumin inhibited H+ extrusion in 2 species of Candida by 42% and 32% in the absence of glucose and by 28% and 18% in the presence of glucose. Respective inhibition of H+ extrusion caused by the MIC of fluconazole was 85% and 89% in the absence of glucose and 61% and 66% in its presence. Ergosterol content decreased by 70% and 53% for the 2 strains following exposure to curcumin at MIC; comparative values for fluconazole at MIC were 93% and 98%. Curcumin and fluconazole decreased proteinase secretion by 49% and 53%, respectively, in C. albicans and by 39% and 46%, respectively, in C. glabrata. In conclusion, curcumin is found to be active against all tested clinical and standard strains but is less effective than fluconazole. Antifungal activity of curcumin might be originating from alteration of membrane-associated properties of ATPase activity, ergosterol biosynthesis, and proteinase secretion.     

大蒜素对白念珠菌形态转换的影响研究

目的探讨大蒜素对白念珠菌形态转换的影响及其作用机制。方法倒置显微镜观察白念珠菌菌丝形成的体外动力学过程;采用CLSI-M27-A3微量液基稀释法检测大蒜素对白念珠菌的最小抑菌浓度(minimum inhibitory concentration,MIC);倒置显微镜观察不同浓度大蒜素对白念珠菌在Spider液体培养基中菌丝形成的影响;qRT-PCR法检测在不同浓度大蒜素作用下白念珠菌菌丝相关基因HWP1、ALS1、EFG1、PDE2表达水平的变化。结果白念珠菌在Spider液体培养基中6 h时出现较长菌丝,24 h后镜下可见大量念珠菌菌丝包裹酵母细胞,紧密交错;大蒜素对白念珠菌的MIC值为25μg/mL;倒置显微镜观察(25~100)μg/mL浓度的大蒜素能明显抑制Spider液体培养基中白念珠菌菌丝的生长;qRT-PCR结果显示,在(25~100)μg/mL浓度的大蒜素作用下,白念珠菌菌丝相关基因表达下调。结论大蒜素能有效抑制白念珠菌的形态转换,其作用机制可能与调节菌丝形成相关基因的表达水平有关。     

大蒜素体外抗白念珠菌生物膜作用的初步研究

目的研究大蒜素对体外白念珠菌生物膜的影响。方法 MTT法评价大蒜素对白念珠菌生物膜形成及细胞黏附的影响;血清芽管计数法评价大蒜素对白念珠菌芽管形成的影响。结果低浓度(4μg/mL)和高浓度(64μg/mL)大蒜素对白念珠菌生物膜形成的抑制率分别为(23.0±1.1)%和(95.6±0.3)%;32μg/mL大蒜素对早期(0h)、中期(12h)及成熟期(48h)生物膜的抑制率分别为(88.5±0.5)%、(63.3±0.8)%和(52.3±1.1)%;与空白对照组相比,不同浓度大蒜素(4～32μg/mL)对培养30min、60min、90min、120min的白念珠菌细胞黏附均有显著抑制作用(P0.05);空白对照组芽管形成率为(91.2±1.6)%,64μg/mL大蒜素组为(2.2±1.2)%。结论大蒜素对体外白念珠菌生物膜有较明显的抑制作用。     

Optically active antifungal azoles: synthesis and antifungal activity of (2R,3S)-2-(2,4-difluorophenyl)-3-(5-[2-[4-aryl-piperazin-1-yl]-ethyl]-tetrazol-2-yl/1-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol

A series of (2R,3S)-2-(2,4-difluorophenyl)-3-(5-[2-[4-aryl-piperazin-1-yl]-ethyl]-tetrazol-2-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol (11a-n) and (2R,3S)-2-(2,4-difluorophenyl)-3-(5-[2-[4-aryl-piperazin-1-yl]-ethyl]-tetrazole-1-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol (12a-n) has been synthesized. The antifungal activity of compounds was evaluated by in vitro agar diffusion and broth dilution assay. Compounds 11d and its positional isomer 12d having 3-trifluoromethyl substitution on the phenyl ring of piperazine demonstrated significant antifungal activity against variety of fungal cultures (Candida spp. C. neoformans and Aspergillus spp.). The compound 12d showed MIC value of 0.12 microg/mL for C. albicans, C. albicans V-01-191A-261 (resistant strain); 0.25 microg/mL for C. tropicalis, C. parapsilosis ATCC 22019 and C. krusei and MIC value of 0.5 microg/mL for C. glabrata, C. krusei ATCC 6258, which is comparable to itraconazole and better than fluconazole. Further, compound 11d showed significant activity (MIC; 0.25-0.5 microg/mL) against Candida spp. and strong anticryptococcal activity (MIC; 0.25 microg/mL) against C. neoformans.     

Identification and susceptibility against fluconazole and albaconazole of 100 yeasts' strains isolated from vaginal discharge

Vulvovaginal candidiasis is a condition that affects a great number of fertile women. It is considered the second cause of genital infection after vaginosis due to GAM complex. Candida albicans is the most frequent isolated species from vaginal discharge. However, sometimes more than one yeast species could be found in the same clinical sample that are more resistant to antifungal drugs. Nowadays, it is necessary to identify properly up to species level the isolated microorganism and to determine the antifungal susceptibility profile. One hundred strains obtained from vaginal discharge of 94 patients suffering acute vulvovaginal candidiasis were studied. The identification of the isolates showed: C. albicans 86%, Candida glabrata 6%, Candida inconspicua 3%, Candida krusei 2% and Candida intermedia, Candida holmii and Trichosporon asahii one case each. Minimal inhibitory concentrations (MIC) of all the yeasts against fluconazole and albaconazole were performed. C. glabrata, C. krusei and C. inconspicua were the most resistant against fluconazole, on the other hand albicans was susceptible to this drug. All the isolates presented MIC against albaconazole much lower than fluconazole.     

In vitro activity of fluconazole and amphotericin B against Candida inconspicua clinical isolates as determined by the time-kill method

Candida inconspicua is an emerging pathogen in immunocompromised patients possessing inherently decreased susceptibility to fluconazole. We determined the MICs and killing activity of fluconazole and amphotericin B against C. inconspicua clinical isolates as well as reference strain C. inconspicua ATCC 16783 for comparison. MICs were determined using the standard broth microdilution method. Killing rates were determined using time-kill methodology at 0.5-16 x MIC fluconazole and amphotericin B concentrations. Fluconazole and amphotericin B MIC values varied between 16-128 mg/l and 0.5-1 mg/l, respectively. In time kill-assays fluconazole showed fungistatic effect at 1-16 x MIC concentrations against all tested strains after 24 h-incubation, but became fungicidal after 48 h at 4-16 x MIC concentrations. The time necessary to achieve fungicidal endpoint at 1 mg/l amphotericin B concentration ranged from 2 to 24 h. Our in vitro results confirm the data that fluconazole is ineffective against C. inconspicua at the fluconazole serum concentration attainable in humans. Amphotericin B due to its rapid killing activity seems to be a good alternative for the treatment of infections caused by C. inconspicua.     

大蒜素对白色念珠菌生长的抑制作用

探讨大蒜素对白色念珠菌生长的抑制作用。以NCCLS方案检测了胡桃楸提取物和伊曲康唑(ICZ)、氟康唑(FCZ)、两性霉素B(AMB)和5-氟胞嘧啶(5-FC)对133株白念珠菌体外生长抑制作用。标准菌株JC1A的M IC结果表明大蒜素提取物对白念珠菌的抗菌作用相当于FCZ,略低于ICZ、AMB和5-FC;临床分离株对ICZ和FCZ的耐药率很高,对大蒜素提取物的耐药率较低。大蒜素提取物对白色念珠菌生长有强力抑制作用。     

Antifungal susceptibility of bloodstream yeasts isolated at a public children's hospital in Brazil: comparison of the Etest and the AFST-EUCAST microdilution method

This study compared the minimum inhibitory concentration (MIC) results from the proposed standard methods of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing (AFST-EUCAST) with the commercial system Etest(R) in the evaluation of susceptibility to flucytosine, fluconazole, itraconazole, voriconazole, and amphotericin B of 136 Candida spp. isolated from the blood of hospitalized children. The results presented a greater agreement among Etest(R) MICs +/-2 log2 dilutions of AFST-EUCAST for fluconazole (98.1% and 96.3%) and voriconazole (100% and 100%) for Candida albicans and Candida parapsilosis. For Candida glabrata, the agreement was greater only for fluconazole (81.8%) and voriconazole (100%). For amphotericin B, the agreement between the methods was low for all species. The agreement percentage among the Etest(R) and AFST-EUCAST susceptibility profiles was high according to the MIC breakpoints recommended by the M27-A2 protocol for the majority of the yeasts, except for fluconazole and itraconazole against Candida tropicalis and for itraconazole against C. glabrata and Candida krusei. According to both methodologies, a great number of Candida spp. isolates showed an in vitro susceptibility to all evaluated antifungal agents. Overall, both procedures can be reliable techniques for susceptibility tests of yeasts, but the assessment of interlaboratory agreement and correlation of MICs by different methods with in vivo response are of great importance.     

Determination of susceptibility/resistance to antifungal drugs of Trichophyton mentagrophytes isolates by a macrodilution method

Onychomycosis is a common adult human mycosis, and dermatophytes of the Trichophyton genera are the most frequently isolated microorganism. Globally, from 3% to 10% of the human population is attacked by ony cho mycosis, and many cases involve toenails. The aim of this work was to determine the minimal inhibitory concentrations (MICs) of antifungal drugs (fluconazole, ketoconazole, itraconazole, terbinafine, and griseofulvin) often used for the treatment of ungueal dermatophytosis caused by Trichophyton mentagrophytes. The MICs were determined by the broth medium macrodilution method. The results showed that activities of terbinafine and itraconazole were significantly higher (MIC <0.007-0.015 microg.mL -1 and MIC = 0.062-1.000 microg.mL -1, respectively). All isolates had reduced susceptibility to fluconazole (MIC = 16 to >64 microg.mL -1). The MICs of ketoconazole and griseofulvin varied among strains, ranging from 0.125 to 2.000 microg.mL -1 for ketoconazole and from 0.25 to 2.00 microg.mL -1 for griseofulvin. These MICs were higher than those of other studies cited, possibly because of differences in culture medium used in the other studies.     

Effect of iron on fluconazole activity against Candida albicans in presence of human serum or monocyte-derived macrophages

Human serum, transferrin, and apotransferrin are known to profoundly inhibit the growth of Candida albicans by iron deprivation. On the other hand, iron overload (iron saturated transferrin) is a serious risk factor for candidiasis in newborn and in leukemic patients. We tested the efficacy of fluconazole and the previously demonstrated synergy of fluconazole and effector cells against C. albicans under iron overload conditions where efficacy might be diminished. We confirm that exogenous iron completely reversed the inhibitory effect of human serum and report that the efficacy of fluconazole against C. albicans was not significantly compromised in a 24 h assay system. Although exogenous iron inhibited fungistatic activity of monocyte-derived macrophages, it did not interfere with the synergistic candidacidal activity of fluconazole and monocyte-derived macrophages. In 72 h assays, where fluconazole had candidacidal activity, exogenous iron did not compromise efficacy of fluconazole, and fluconazole activity was often increased. These in vitro results suggest that effectiveness of fluconazole therapy would not be compromised in iron overload situations in vivo. This revised version was published online in August 2006 with corrections to the Cover Date.     

Determination of antifungal activities in serum samples from mice treated with different antifungal drugs allows detection of an active metabolite of itraconazole

To establish an in vitro method of predicting in vivo efficacy of antifungal drugs against Candida albicans and Aspergillus fumigatus, the antifungal activities of fluconazole, itraconazole, and amphotericin B were determined in mouse serum. The minimum inhibitory concentration (MIC) of each drug was measured using mouse serum as a diluent. For C. albicans, the assay endpoint of azoles was defined as inhibition of mycelial extension (mMIC) and for A. fumigatus, as no growth (MIC). The MICs of amphotericin B for both pathogens were defined as the MIC at which no mycelial growth occurred. Serum MIC or mMIC determinations were then used to estimate the concentration of the drugs in serum of mice treated with antifungal drugs by multiplying the antifungal titer of the serum samples by the serum (m)MIC. The serum drug concentrations were also determined by HPLC. The serum concentrations estimated microbiologically showed good agreement with those determined by HPLC, except for itraconazole. Analysis of the serum samples from itraconazole-treated mice by a sensitive bioautography revealed the presence of additional spots, not seen in control samples of itraconazole. The bioautography assay demonstrated that the additional material detected in serum from mice treated with itraconazole was an active metabolite of itraconazole. The data showed that the apparent reduction in the itraconazole serum concentration as determined by HPLC was the result of the formation of an active metabolite, and that the use of a microbiological method to measure serum concentrations of drugs can provide a method for prediction of in vivo efficacy of antifungal drugs.     

In Vitro Investigation of Antifungal Activity of Allicin Alone and in Combination with Azoles Against <Emphasis Type="Italic">Candida</Emphasis> Species

Candidiasis is a term describing infections by yeasts from the genus Candida, and the type of infection encompassed by candidiasis ranges from superficial to systemic. Treatment of such infections often requires antifungals such as the azoles, but increased use of these drugs has led to selection of yeasts with increased resistance to these drugs. In this study, we used allicin, an allyl sulfur derivative of garlic, to demonstrate both its intrinsic antifungal activity and its synergy with the azoles, in the treatment of these yeasts in vitro. In this study, the MIC₅₀ and MIC₉₀ of allicin alone against six Candida spp. ranged from 0.05 to 25 μg/ml. However, when allicin was used in combination with fluconazole or ketoconazole, the MICs were decreased in some isolates. Our results demonstrated the existing synergistic effect between allicin and azoles in some of the Candida spp. such as C. albicans, C. glabrata and C. tropicalis, but synergy was not demonstrated in the majority of Candida spp. tested. Nonetheless, In vivo testing needs to be performed to support these findings.