Effect of amphotericin B on the enzyme system of Candida albicans]

The mechanism of amphotericin B action was studied with the aid of cytochemical methods providing determination of the activity of the 4 main enzymes characterizing the cell energetics, i. e. succinate dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase and glucose-6-phosphate dehydrogenase inside the cell. With an increase in the concentration of amphotericin B in the medium the activity of all the 4 enzymes decreased, the percentage of the inactive cells being regularly increased. Changes in the fermentative activity of C. albicans as dependent on the incubation time with the antibiotic were studied. Only the respiration activity decreased in 2 hours. As a result of a 4-hour exposure to the polyen in the cells of C. albicans the activity of the lactic acid fermentation, respiration through succinate dehydrogenase and activity of the pentose shunt decreased 1.5--2 times. In 24 hours of incubation the activity of the above decreased 80--90 per cent as compared to the activity of the initial culture.     

Characteristics of a strain of C. albicans resistant to polyene antibiotics]

It was found that a resistant strain R2 of C. albicans obtained as a result of passages on media containing increasing concentrations of amphotericin B differed from the initial strain by its lower pathogenicity. Treatment of the infection caused by the resistant strain on modeling of candidiasis in mice was not successful. The decrease in the average life span of the mice infected with the resistant strain R2 and treated with amphotericin B was lower than that in the control animals and such indices of the disease as the levels of the kidney dissemination and the cell vegetation even increased under the effect of amphotericin B. The results of the study suggest that the resistant strain R2 of C. albicans depend on amphotericin B in the host. The data obtained emphasize the necessity of determinining the antibiotic sensitivity of C. albicans strains isolated from patients.     

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.     

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.     

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.     

Quantitative and qualitative analyses of the cell death process in Candida albicans treated by antifungal agents

The death process of Candida albicans was investigated after treatment with the antifungal agents flucytosine and amphotericin B by assessing morphological and biophysical properties associated with cell death. C. albicans was treated varying time periods (from 6 to 48 hours) and examined by scanning electron microscopy (SEM) and atomic force microscopy (AFM). SEM and AFM images clearly showed changes in morphology and biophysical properties. After drug treatment, the membrane of C. albicans was perforated, deformed, and shrunken. Compared to the control, C. albicans treated with flucytosine was softer and initially showed a greater adhesive force. Conversely, C. albicans treated with amphotericin B was harder and had a lower adhesive force. In both cases, the surface roughness increased as the treatment time increased. The relationships between morphological changes and the drugs were observed by AFM clearly; the surface of C. albicans treated with flucytosine underwent membrane collapse, expansion of holes, and shrinkage, while the membranes of cells treated with amphotericin B peeled off. According to these observations, the death process of C. albicans was divided into 4 phases, CDP(0), CDP(1), CDP(2), and CDP(4), which were determined based on morphological changes. Our results could be employed to further investigate the antifungal activity of compounds derived from natural sources.     

Experimental murine mycotic mastitis: a sensitive and lenient model for studies of antifungal chemotherapy

The murine model of mycotic mastitis was used to study the efficacy of amphotericin B (AmB). Twenty-four BALB/cJ mice at the fifth day of lactation were anesthetized and inoculated through the teat canal (two glands) with 50 microl suspension containing 5.0 x 10(7) cfu ml(-1) Candida albicans blastospores. Mice were randomly divided into two groups: untreated controls and AmB treated. Animals were euthanized 3 and 6 days after infection and treatment (4 mg kg(-1) per day intraperitoneally). The fungal burden of the mammary gland was determined by quantitative cultures. The number of C. albicans cells recovered from mammary gland homogenates were significantly lower in the AmB treated animals (both 3 and 6 days post-infection) than in the untreated controls (P<0.007 and P<0.003, respectively). The mammary glands of all untreated control animals showed marked neutrophilic infiltration, severe necrosis, and presence of blastospores, hyphae and pseudohyphae. In contrast, 10 of 12 animals treated with AmB showed only a mild neutrophilic infiltration which was restricted to alveoli and excretory ducts. All extra-mammary organs were free of infection in both groups. The results demonstrate that the murine mycotic mastitis model is suitable for investigations of new antifungal compounds. In addition, this model is more lenient than the systemic candidiasis models.     

Comparative efficacy of fluconazole and amphotericin B in the parenteral treatment of experimental paracoccidioidomycosis in the rat

Patients with severe and complicated paracoccidioidomycosis are treated with amphotericin B by the intravenous route. Fluconazole is active in vitro against Paracoccidioides brasiliensis and can also be administered intravenously, but few clinical or experimental data are available about its action against the infection caused by this fungus. In the present study, the efficacy of fluconazole andamphotericin B was assessed comparatively in rats inoculated parenterally with P. brasiliensis. The treatment was performed 3 times a week for 4 weeks starting one week after infection. Fluconazole administered intraperitoneally (14 mg/kg bodyweight/dose) was more effective (P > 0.001)than amphotericin B (2 mg/kg body weight/dose) in reducing the number of colony forming units in the lungs and spleen. When administered intravenously at the dose of 3 mg/kg body weight, fluconazole was as effective as amphotericin B (0.8 mg/kg body weight) in reducing the pulmonary fungal burden. Under these conditions, the rats treated with fluconazole had a smaller number of colony forming units than untreated animals (P > 0.001), but amphotericin B was more effective than fluconazole in reducing spleen infection (P > 0.005). Except for this result obtained with a low dose, fluconazole showed an antifungal action equal to or higher than that of amphotericin B. The activity of fluconazole at doses equivalent to those used for human treatment suggests that this antifungal agent may be an alternative to amphotericin B for the early intravenous treatment of patients with paracoccidioidomycosis. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Chlamydospore-like cells of Candida albicans in the gastrointestinal tract of infected, immunocompromised mice

We have demonstrated in a previously described murine model of gastrointestinal (GI) and systemic candidiasis that the antifungal angent cilofungin was efficacious in clearing infection of body organs when administered subcutaneously by infusion, but permitted large numbers of Candida albicans in the GI tract to persist. Yeast and hyphae in these animals were associated primarily with the stratified squamous epithelium of the stomach. Administration of immunocompromising drugs (cyclophosphamide plus cortisone acetate) to animals with persistent GI infection resulted in relapse of systemic candidiasis. Histological examination of the gastric mucosa revealed invasive hyphal elements and yeast as well as multiple chlamydospore-like cells. Comparative histochemical and electron-microscopic examinations of these latter cells produced in host tissue and chlamydospores formed in vitro were conducted. The results suggested that similarities in wall and cytoplasmic composition and ultrastructure exist between these in vivo and in vitro produced C. albicans cells. Exposure of C. albicans to cyclophosphamide during in vitro growth resulted in stimulation of chlamydospore production. No significant effect of cortisone acetate on C. albicans morphogenesis was detected. The murine model used in this study permits investigation of the formation of chlamydospore-like cells of C. albicans during early stages of fungal invasion of cyclophosphamide-treated mice, and of the possible influence of these cells on immunological response of the host to persistent candidiasis of the GI tract.     

Clinical strains of Candida albicans express the surface antigen glyceraldehyde 3-phosphate dehydrogenase in vitro and in infected tissues

We have previously described the presence of an enzymatically active form of glyceraldehyde 3-phosphate-dehydrogenase (GAPDH) in the cell surface of Candida albicans ATCC 26555 which is also a fibronectin and laminin binding protein. Immunohistochemical analysis of tissue sections from patients with disseminated candidiasis with a polyclonal antiserum to GAPDH from C. albicans (PAb anti-CA-GAPDH) revealed that the enzyme is expressed at the surface of fungal cells in infected tissues. The same PAb detected the presence of GAPDH species, with a molecular mass of approximately 33 kDa, in cell wall extracts obtained from clinical isolates of the fungus. These cell surface-bound GAPDH moieties exhibited a dose-dependent dehydrogenase activity. These results indicate that this cell surface-bound GAPDH plays a role during infection probably contributing to the attachment of fungal cells to host tissues.     

The effects of amphotericin B on the interaction of Candida albicans with fibroblast cultures

Sublethal amounts of amphotericin B inhibited the interaction of Candida albicans with cultured fibroblasts. Different C. albicans clinical isolates exhibited varying degrees of sensitivity to the drug, but those isolates that were the most infective in control cultures appeared to be the most resistant to amphotericin B mediated infection inhibition. Although amphotericin B inhibited germ tube formation at the sublethal concentration of 0.3 microgram/mL, lower concentrations inhibited infection without preventing germination. The extent of this latter activity varied with the isolate and amphotericin B concentration and appeared to be related to sublethal effects on germinated yeasts. While amphotericin B effectively prevented new fibroblast infection, it did not dissociate those yeasts which had established an infection before its addition.     

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.     

Species distribution, antifungal susceptibility and clonal relatedness of Candida isolates from patients in neonatal and pediatric intensive care units at a medical center in Turkey

The aim of this study was to assess species distribution, antifungal susceptibility and clonal relationships among Candida strains isolated from a group of pediatric/neonatal intensive care (PICU/NICU) patients that had a very high mortality rate (76%). The cases of 21 patients (19 with candidemia, 2 with Candida meningitides) treated over a 1-year period in a Turkish hospital PICU and NICU were retrospectively analyzed. Twenty-eight Candida isolates were detected from blood (20), cerebrospinal fluid (CSF) (2) and other specimens (6). Candida species were identified using the API ID 32C System. Susceptibility testing was done (all 28 isolates) for amphotericin B, fluconazole and itraconazole using the broth microdilution method. Arbitrarily primed polymerase chain reaction (AP-PCR) was used for molecular typing of the 3 most common ones; C. albicans (15), C. parapsilosis (6), and C. pelliculosa (4). Electrophoretic karyotyping (EK) was done to check clonal identity obtained by AP-PCR. Of the 20 blood isolates, 8 (40%) were C. albicans, 12 (60%) were non-albicans Candida, and one of the 2 CSF isolates was C. albicans. The overall species distribution was as follows: 15 C. albicans isolates, 6 C. parapsilosis isolates, 4 C. pelliculosa isolates, 2 C. famata isolates and 1 C tropicalis isolate. Amphotericin B had the best antifungal activity with a MIC90 of 0.125 microg/ml, and the rates of susceptibility to fluconazole and itraconazole were 93% and 82%, respectively. AP-PCR revealed 11 genotypes (4 were identical pairs, 7 were distinct) among the 15 C. albicans isolates, 2 genotypes (5 were classified in the same type) among the 6 C. parapsilosis isolates, and 4 separate genotypes for the 4 C. pelliculosa isolates. Karyotyping results correlated well with the AP-PCR findings. As indicated in the previous research, our results confirmed that non-albicans Candida species have become more frequently causative agents for invasive fungal infections in the ICU. Transmission of C. albicans and C. pelliculosa was relatively low, but transmission of C. parapsilosis was high, suggesting that more effective control and very strict treatment protocols are needed for patients having high mortality and invasive fungal infection in ICU.     

重症监护病房白念珠菌耐药性8年变化趋势

目的调查上海长征医院重症监护室(ICU)近8 a中临床分离白念珠菌的耐药性变化,为临床治疗提供参考。方法上海长征医院ICU 2002～2009年从414例患者中首次分离出414株白念珠菌,对其中277株进行药敏试验。采用Cox-Stuart趋势检验回顾性分析临床分离真菌中白念珠菌所占比例变化趋势和白念珠菌对常用抗真菌药物耐药率的变化趋势。结果 2002～2009年间,上海长征医院ICU白念珠菌分离株数从2002年的34株增加至2009年的92株,但白念珠菌占总真菌分离株数的百分比维持在34.6%～55.7%,P=0.03。白念珠菌对于5-氟胞嘧啶和两性霉素B平均耐药率分别为4.0%和0.7%,对其他常用抗真菌药的耐药率依次为咪康唑47.0%、酮康唑10.8%、伊曲康唑19.9%、特比萘芬42.6%、氟康唑14.6%及伏立康唑13.0%。白念珠菌对5-氟胞嘧啶和两性霉素B和伊曲康唑耐药率的8年变化无统计学差异。结论上海长征医院ICU近8 a来白念珠菌仍然为临床较为常见的真菌分离株,但白念珠菌占总分离株数的百分比有逐渐减少的趋势。白念珠菌对常用抗真菌药物耐药性均无明显变化趋势。     

Effects of imidazole- and triazole-derivative antifungal compounds on the growth and morphological development of Candida albicans hyphae

Six azole-derivative antifungal compounds affected several aspects of Candida albicans hyphal development with only a relatively small degree of inhibition of growth rate, measured in terms of ATP concentration, whereas amphotericin B and 5-fluorocytosine affected morphology only when they also substantially inhibited fungal growth rate. At 10(-8) M, all the azoles tested inhibited branch formation by C. albicans hyphae. At 10(-7) M and higher concentrations, clotrimazole and miconazole strongly suppressed emergence of new hyphal outgrowths from parent yeast cells, whereas ICI 153066 and itraconazole had little effect on this phenomenon and ketoconazole and tioconazole had intermediate effects. At the highest concentrations tested (10(-5) M) hyphal development was ultimately arrested by the azole compounds and the fungus grew predominantly in the form of budding yeast cells; however, none of the azole antifungals prevented initial emergence of an apparently normal germ tube. The antifungals only exerted their morphological effects when they were present in the culture medium: removal of the compounds after exposure of C. albicans to them led to reversion to normal growth.     

Modern antifungals in therapy of nosocomial mycosis in oncologic patients

Rational position of voriconazole in the treatment of oncologic inpatients was shown and the criteria of its use in the algorithms of the therapy and prophylaxis of nosocomial fungal infections were developed. The clinical trial enrolled 50 patients with oncologic pathologies. The patients were divided into two groups of possible invasive candidiasis risk. The patients of one group were treated with fluconazole (Diflucan) and those of the other group were treated with voriconazole (Vifend). The spectrum of the hospital fungal flora was determined and susceptibility of 310 clinically important opportunistic fungi was investigated. All the isolates of Candida albicans and C.tropicalis were susceptible to amphotericin B, fluconazole and voriconazole and 79 and 50% of the isolates were susceptible to intraconazole respectively. As for the C.krusei isolates, 67% was susceptible to amphotericin B, 50% was susceptible to fluconazole, 100% was susceptible to voriconazole and none of the strains was susceptible to intraconazole. By the clinical efficacy voriconazole was superior to fluconazole and comparable with amphotericin B, while superior to it by the number of the side effects and by the cost of the treatment course. It was concluded that voriconazole should be considered as the main agent in the antifungal therapy of oncologic patients.     

Inhibition of Candida albicans extracellular enzyme activity by selected natural substances and their application in Candida infection

Extracellular enzymes secreted by Candida albicans are claimed to be virulence factors responsible for penetration of the yeast into host cells. Substances able to inhibit lipolytic and proteinase activities of the fungus might be of therapeutic use in some pathologic conditions caused by C. albicans. In the present work, we have tested the influence of the flavonoid compounds apigenin and kaempferol, the indole alkaloid ibogaine, and the protoberberine alkaloid berberine on the in vitro enzyme activity of C. albicans. The substances showed complex suppressive effects concerning the processes of adherence to epithelial cells, secreted aspartyl proteinase activity, and the rate of cell wall protein glycosylation. Apigenin and kaempferol were administered in systemic C. albicans infection, demonstrating an increased number of survivors by kaempferol. The application of apigenin, kaempferol, ibogaine, and berberine in cutaneous infection suppressed the symptoms and accelerated elimination of the yeast from the site of inoculation.     

THIS ARTICLE HAS BEEN RETRACTEDHamycin treatment of candidiasis in normal and diabetic rats

Hamycin, a heptaene antifungal antibiotic was compared with amphotericin B in the treatment of established systemic infection with Candida albicans in normal and diabetic rats. In normal rats, orally administered hamycin at 10 mg kg(-1) per day for 7 days reduced Candida colony counts in the kidneys and livers as well as amphotericin B did and was nearly as effective as amphotericin B in a 21-day treatment trial. There was no further reduction in Candida colony counts when normal rats were treated with hamycin at 25 mg kg(-1) twice a day for 7 days. In streptozotocin induced diabetic rats, hamycin at 20 mg kg(-1) per day for either 7 or 21 days compared favourably with amphotericin B in efficacy. Results of the present study suggest that oral hamycin may be useful in the treatment of established disseminated candidiasis in normal as well as diabetic hosts.     

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.