Quantitative and qualitative analysis of the antifungal activity of allicin alone and in combination with antifungal drugs

The antifungal activity of allicin and its synergistic effects with the antifungal agents flucytosine and amphotericin B (AmB) were investigated in Candida albicans (C. albicans). C. albicans was treated with different conditions of compounds alone and in combination (allicin, AmB, flucytosine, allicin + AmB, allicin + flucytosine, allicin + AmB + flucytosine). After a 24-hour treatment, cells were examined by scanning electron microscopy (SEM) and atomic force microscopy (AFM) to measure morphological and biophysical properties associated with cell death. The clearing assay was conducted to confirm the effects of allicin. The viability of C. albicans treated by allicin alone or with one antifungal drug (AmB, flucytosine) in addition was more than 40% after a 24-hr treatment, but the viability of groups treated with combinations of more than two drugs was less than 32%. When the cells were treated with allicin alone or one type of drug, the morphology of the cells did not change noticeably, but when cells were treated with combinations of drugs, there were noticeable morphological changes. In particular, cells treated with allicin + AmB had significant membrane damage (burst or collapsed membranes). Classification of cells according to their cell death phase (CDP) allowed us to determine the relationship between cell viability and treatment conditions in detail. The adhesive force was decreased by the treatment in all groups compare to the control. Cells treated with AmB + allicin had a greater adhesive force than cells treated with AmB alone because of the secretion of molecules due to collapsed membranes. All cells treated with allicin or drugs were softer than the control cells. These results suggest that allicin can reduce MIC of AmB while keeping the same efficacy.     

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...     

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。     

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.     

Disseminated candidiasis: evidence of a distinctive syndrome in heroin abusers.

Seven young men developed similar manifestations of disseminated candidiasis after a single episode of intravenous heroin abuse. Sequential development of lesions of the eye, skin, and bone or costal cartilage was noted within 10 days after injection. Skin lesions were confined to the scalp and other hair bearing areas. Candida albicans was cultured readily from affected skin and costal cartilage. Histological examination of scalp biopsy specimens showed infiltration of hair follicles with chronic inflammatory cells and C albicans. Pseudohyphas of C albicans were also identified in and around hair shafts. The skin, skeletal, and small eye lesions resolved on systemic treatment with 1 g amphotericin B plus flucytosine. Pars plana vitrectomy plus local instillation of amphotericin B cured progressive chorioretinitis. These features may represent a distinctive syndrome of disseminated candidiasis in heroin abusers. Systemic antifungal treatment is curative in most cases.     

Comparative evaluation of standard dilution method and commercial kit for frozen plate antifungal susceptibility testing of yeasts using 200 clinical isolates

A comparative evaluation of standard microdilution methods and a commercial kit for frozen plate antifungal susceptibility testing of yeasts was performed using amphotericin B, flucytosine, fluconazole, miconazole, and itraconazole on 200 yeast isolates. The isolates included 100 strains of Candida albicans, eight of C. tropicalis, twelve of C. parapsilosis, eight of C. glabrata, five of Cryptococcus neoformans, thirteen of Trichosporon asahii, and 54 other strains of seven other species of ascomycotic yeasts. Microdilution testing was performed according to the standard method for antifungal susceptibility testing published by the Japanese Society for Medical Mycology (JSMM), which are a modification of the method developed by the National Committee for Clinical Laboratory Standards (NCCLS) M27-P. The commercial kit was prepared according to the manufacturer's instructions. The degree of agreement within +/-1 dilution for 200 clinical isolates against five antifungal agents was excellent with values for amphotericin B, flucytosine, fluconazole, miconazole, and itraconazole of 100%, 99.0%, 97.5%, 97.0%, and 97.0%, respectively. Overall, the frozen plate antifungal susceptibility testing kit provided convenient and reproducible results comparable to those obtained with the JSMM standard method.     

In vitro resistance to fluconazole and itraconazole in clinical isolates of Candida spp and Cryptococcus neoformans

An in vitro susceptibility testing of 181 strains of six species of Candida and 21 strains of Cryptococcus neoformans was carried out in order to investigate the resistance to new antifungal drugs. We have studied clinical isolates from 200 different patients of Hospital del Mar (Barcelona) and Hospital La Inmaculada (Almería). An agar diffusion method (NeoSensitabs, Rosco, Taastrup, Denmark), was employed with fluconazole, itraconazole, and reference drugs amphotericin B, flucytosine, tioconazole and ketoconazole. A high level of susceptibility was found for amphotericin B in C. neoformans strains while 19% of them were resistant to flucytosine. All the strains of C. neoformans and Candida guilliermondii were susceptible to the new azoles derivatives and also Candida parapsilosis and Candida albicans had a great susceptibility to this antifungals. A greater level of resistance was found for Candida krusei, Candida tropicalis and Candida glabrata to fluconazole, itraconazole and ketoconazole, but resistance to fluconazole and itraconazole is not always linked because ten resistant strains for fluconazole were susceptible to itraconazole, and two other resistant to itraconazole were susceptible to fluconazole.     

In vitro activity of amphotericin B and anidulafungin against Candida spp. biofilms

Invasive infections caused by Candida spp. are increasing worldwide and are becoming an important cause of morbidity and mortality in immunocompromised patients. A large number of manifestations of candidiasis are associated with the formation of biofilms on inert or biological surfaces. Candida spp. biofilms are recalcitrant to treatment with conventional antifungal therapies. The aim of this study was dual 1) to determine the prevalence of biofilm producers among clinical isolates from catheter (16 C. albicans ) and blood culture (2 C. albicans and 30 C. tropicalis), and 2) to determine the activity of amphotericin B and anidulafungin against C. albicans and C. tropicalis biofilms of 24 and 48 hours of maturation. Biofilms were developed using a 96-well microtitre plate model and production and activity of antifungal agents against biofilms were determined by the tetrazolium (XTT) reduction assay. Of catheter and blood isolates, 62.5 and 56.25%, respectively, produced biofilms. By species, 68.42% of C. albicans and 53.33% of C. tropicalis were biofilm producers. C. albicans biofilms showed more resistance to amphotericin B and anidulafungin than their planktonic counterparts. Complete killing of biofilms was never achieved, even at the highest concentrations of the drugs tested. Anidulafungin displayed more activity than amphotericin B against C. albicans biofilms of 24 hours of maturation (GM MIC 0.354 vs. 0.686 microg/ml), but against C. tropicalis biofilms amphotericin B was more active (GM MIC 11.285 vs. 0.476 microg/ml). In contrast, against biofilms with 48 hours maturation, amphotericin B was more active against both species.     

In Candida albicans, resistance to flucytosine and terbinafine is linked to MAT locus homozygosity and multilocus sequence typing clade 1

A panel of 637 isolates of Candida albicans that had been typed by multilocus sequence typing (MLST) and tested for susceptibility to amphotericin B, caspofungin, fluconazole, flucytosine, itraconazole, ketoconazole, miconazole, terbinafine and voriconazole was the material for a statistical analysis of possible associations between antifungal susceptibility and other properties. For terbinafine and flucytosine, the greatest proportion of low-susceptibility isolates, judged by two resistance breakpoints, was found in MLST clade 1 and among isolates homozygous at the MAT locus, although only three isolates showed cross-resistance to the two agents. Most instances of low susceptibility to azoles, flucytosine and terbinafine were among oropharyngeal isolates from HIV-positive individuals. Statistically significant correlations were found between terbinafine and azole minimal inhibitory concentrations (MICs), while correlations between flucytosine MICs and azole MICs were less strong. It is concluded that a common regulatory mechanism may operate to generate resistance to the two classes of agent that inhibit ergosterol biosynthesis, terbinafine and the azoles, but that flucytosine resistance, although still commonly associated with MAT homozygosity, is differently regulated.     

白念珠菌临床分离调查及基因分型研究

目的 了解白念珠菌临床分离情况,并探讨其药敏结果与基因分型的相关性.方法 回顾性分析本院2011年3～11月间临床分离白念珠菌分布及耐药性;随机选取232株,采用PCR方法扩增白念珠菌25S rDNA基因内含子区进行基因分型研究;采用ATB真菌药敏试剂条进行药敏分析;统计分析药敏结果与基因分型的相关性.结果 期间共检出酵母样真菌973例,占病原菌阳性样本数比率为15.7％ (973/6196);其中分离白念珠菌562株,占58％ (562/973),主要分布科室为呼吸科(39.1％)、老年科(13.2％)、ICU(7.7％)、神经内科(7.5％)、免疫科(6.0％)以及其他科室(26.5％);标本类型以下呼吸道为主(81.7％),其次为尿路(9.4％)、血液(1.8％)等.对氟胞嘧啶、两性霉素B、氟康唑、伊曲康唑及伏立康唑的耐药率分别为0.9％、0％、1.4％、1.6％和1.1％.随机选取的232株白念珠菌经PCR方法可分为3型:A型125株,B型96株,C型11株.各型在5种药物的耐药性上并无差异.结论 临床分离酵母样真菌以白念珠菌为主,感染部位以下呼吸道为主;临床分离株对5种抗真菌药物敏感度较高,主要基因型为A和B型,不同基因分型间药敏结果并无统计学差异.     

Occurrence and the sensitivity to drugs of fungi other than Candida albicans isolated from the vagina

Frequency of occurrence of fungal species distinct from C. albicans isolated from vaginal mucosa and their sensitivity to antimycotic chemotherapeutics was determined. Material consisted of 452 fungal strains isolated from vagina from patients suffering from afflictions within genital area. Fungal strains isolated belonged to 13 genera. Fungi distinct from C. albicans constituted 27.1% of all the strains. Fungi the most frequently isolated from vagina belonged to following genera: T. glabrata 35.2% C. krusei 18.4% C. pseudotropicalis 15.2% S. cerevisiae 10.4%. In the majority of cases of vaginal infections caused by fungi distinct from C. albicans, Lactobacillus sp. was present and normal pH values of vaginal content 3/4 with variable number of leucocytes were observed. Evaluation of sensitivity to antimycotic drugs of fungal strains was performed by agar dilution technique. In this study the following chemotherapeutics were assayed: nystatin, pimaricin, amphotericin B, flucytosine, clotrimazole, miconazole and ketoconazole. It is worth to underline resistance of T. glabrata and S. cerevisiae to clotrimazole and ketoconazole. Moreover, resistance of strains belonging to genera C. krusei and C. pseudotropicalis to amphotericin B and C. krusei strains to nystatin and flucytosine was noted.     

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.     

Evaluation of Systemic Antifungal Agents in X-Irradiated Mice

The effect of X irradiation on the survival time of animals experimentally infected with pathogenic fungi was studied, and the activity of antifungal agents in pre-irradiated hosts was evaluated. A 24-hr preinfection dose of X irradiation decreased the survival time of mice infected with Cryptococcus neoformans and Histoplasma capsulatum to a greater extent than Candida albicans or Blastomyces dermatitidis infections. Exposure to 400 r caused a significant reduction in the variation (S(2)) survival time of C. albicans or H. capsulatum mouse infections. A single 100-mg/kg dose of 5-fluorocytosine or amphotericin B administered within 24 hr postinfection significantly extended the survival time of mice infected with C. albicans. Delayed treatment with amphotericin B was effective against C. neoformans infections. Four 50-mg/kg doses of 5-fluorocytosine were more effective than a single 200-mg/kg dose against C. neoformans infections. A single dose of amphotericin B provided significant protection when administered 48 hr postinfection against B. dermatitidis in preirradiated mice. A single dose of saramycetin 48 hr postinfection was highly effective against H. capsulatum mouse infections. A 100-mg/kg dose of amphotericin B was only effective against this fungal pathogen when administered within 8 hr postinfection. In vivo activity of the antifungal agents studied was detected within 8 to 14 days. The relative in vivo activity of several antifungal agents indicated the importance of considering their individual pharmacological properties for optimum effectiveness. The experimental model used in this study should be useful for the detection and for the preclinical evaluation of new antifungal agents.     

Effect of mycolase and amphotericin B on Candida albicans and Candida pseudotropicalis in vitro and in vivo

A mixture of enzymes (mycolase) capable of lysing yeast cell walls was prepared from culture filtrates of Physarum polycephalum. The enzymes present in mycolase included chitinase, beta-1,3-glucanases and exo-glycosidases. The pH optima of these enzymes were in the range 3.5-5.0 and they had low activities at pH 7.0. Mycolase produced spheroplasts from Candida pseudotropicalis and, unlike commercial enzyme preparations such as L1, chitinase, beta, 1,3-glucanase and beta-glucosidase, had some candicidal activity in vitro against C. pseudotropicalis and C. albicans. Mycolase potentiated the antifungal activity of amphotericin B against C. pseudotropicalis grown in shake flask culture but did not potentiate the antifungal activity of the antibiotic against similar cultures of C. albicans; indeed antagonism between mycolase and amphotericin B was sometimes observed with the latter yeast. Mycolase caused an approximately two-fold increase in the total and viable counts of cultures of C. albicans inoculated with stationary phase cells. These increases, which were observed within about 30 min, were attributed to mycolase inducing the premature release of viable buds from 'lag' phase cells. Mycolase also increased the rate at which C. albicans formed germ tubes when the yeast was cultured in a medium containing serum. Mycolase alone or in combination with amphotericin B did not appreciably enhance phagocytosis or intracellular killing of the yeasts by unstimulated mouse peritoneal macrophages. Studies on mice infected systemically with C. albicans showed that mycolase only slightly enhanced amphotericin B therapy.     

Phenotype and genotype of Candida albicans strains isolated from pregnant women with recurrent vaginitis

Fourteen out of 80 pregnant women receiving prenatal care presented signs and symptoms of recurrent vaginal candidiasis. Candida albicans strains were isolated from 12 patients (85.7%), and these were submitted to morphotyping (morphological characteristics of the colony), antifungal typing (pattern of sensitivity to amphotericin B, 5-fluorcytosine, myconazole, ketoconazole and fluconazole) and genotyping (electrophoretic migration of DNA fragments digested with EcoRI and HinfI). Alteration of morphotype and antifungal type was observed in 50% of the patients, but the genotype of the strains isolated from the same patients at different times was identical in all subjects. The predominant morphotypes presented continuous fringes and the basic changes observed among antifungal types was the emergence of strains resistant to myconazole, which was the drug used for the treatment of the first episode of vaginitis. We conclude that recurrent vaginal candidiasis is caused by the persistence of a single yeast genotype that undergoes morphological and behavioral changes in the presence of antifungal agents due to the selective pressure to which it is submitted. This revised version was published online in August 2006 with corrections to the Cover Date.     

Resveratrol lacks antifungal activity against Candida albicans

The putative candicidal activity of resveratrol is currently a matter of controversy. Here, the antifungal activity as well as the antioxidant response of resveratrol against Candida albicans, have been tested in a set of strains with a well-established genetic background At the doses usually employed in antifungal tests (10-40 μg/ml), resveratrol has no effect on the exponential growth of the C. albicans CAI.4 strain, a tenfold increase (400 μg/ml) was required in order to record a certain degree of cell killing, which was negligible in comparison with the strong antifungal effect caused by the addition of amphotericin B (5 μg/ml). An identical pattern was recorded in the prototrophic strains of C. albicans SC5314 and RM-100, whereas the oxidative sensitive trehalose-deficient mutant (tps1/tps1 strain) was totally refractory to the presence of resveratrol. In turn, the serum-induced yeast-to-hypha transition remained unaffected upon addition of different concentrations of resveratrol. Determination of endogenous trehalose and catalase activity, two antioxidant markers in C. albicans; revealed no significant changes in their basal contents induced by resveratrol. Collectively, our results seem to dismiss a main antifungal role as well as the therapeutic application of resveratrol against the infections caused by C. albicans.     

Antimycotic therapy of experimental infections caused by dematiaceous fungi

Experimental infections of mice with Wangiella dermatitidis and Fonsecaea pedrosoi provided a model for evaluating new antifungal agents or new combination therapy. In our models flucytosine exerted a dose-related therapeutic effect on the acute and on the more chronic infection. In the acute Wangiella infection amphotericin B also showed therapeutic activity whereas in the Fonsecaea model the effect was weak. The azole derivative ICI 153066 was the most efficacious drug in the Wangiella model whereas ketoconazole was inactive. The effect on colony-forming units of fungi in the brain was stronger with all drugs tested than the effect on survival time. Combination therapy with flucytosine + amphotericin B showed reproducible potentiating effects whereas the combination of flucytosine + ketoconazole was only additive and amphotericin B + ketoconazole showed no synergistic effect.     

Modification of the fluorescein diacetate assay for screening of antifungal agents against Candida albicans: comparison with the NCCLS methods

A modified fluorescein diacetate (FDA) assay has been compared with standard NCCLS broth macrodilution and broth microdilution methods for the detection of antifungal activity. The FDA assay was performed in a medium containing bacteriological peptone, NaCl, yeast extract and glucose (0.2%, 0.1%, 0.1% and 1% w/v, respectively) and buffered with 10 mM BES buffer. The MICs of amphotericin B, fluconazole, miconazole and flucytosine (representing three major classes of antifungal agents) obtained by the three methods were compared. The results obtained with the FDA assays correlated well with the NCCLS macrodilution method for MICs of amphotericin B, miconazole and fluconazole, but not for flucytosine. However, the MIC values of flucytosine obtained with the FDA assay were well within the quality control range for the two reference strains recommended by the NCCLS. The FDA assay described is an attractive alternative to the NCCLS methods for screening for antifungal agents, with the added advantage of objectivity of fluorescence measurement.     

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。     

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.