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。     

A unique fungal two-component system regulates stress responses, drug sensitivity, sexual development, and virulence of Cryptococcus neoformans

The stress-activated mitogen-activated protein kinase (MAPK) pathway is widely used by eukaryotic organisms as a central conduit via which cellular responses to the environment effect growth and differentiation. The basidiomycetous human fungal pathogen Cryptococcus neoformans uniquely uses the stress-activated Pbs2-Hog1 MAPK system to govern a plethora of cellular events, including stress responses, drug sensitivity, sexual reproduction, and virulence. Here, we characterized a fungal "two-component" system that controls these fundamental cellular functions via the Pbs2-Hog1 MAPK cascade. A typical response regulator, Ssk1, modulated all Hog1-dependent phenotypes by controlling Hog1 phosphorylation, indicating that Ssk1 is the major upstream signaling component of the Pbs2-Hog1 pathway. A second response regulator, Skn7, governs sensitivity to Na+ ions and the antifungal agent fludioxonil, negatively controls melanin production, and functions independently of Hog1 regulation. To control these response regulators, C. neoformans uses multiple sensor kinases, including two-component-like (Tco) 1 and Tco2. Tco1 and Tco2 play shared and distinct roles in stress responses and drug sensitivity through the Hog1 MAPK system. Furthermore, each sensor kinase mediates unique cellular functions for virulence and morphological differentiation. Our findings highlight unique adaptations of this global two-component MAPK signaling cascade in a ubiquitous human fungal pathogen.     

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.     

Cryptococcus neoformans: The Model Organism for Yeast Antifungal Drug Susceptibility Testing

We describe an approach to antifungal susceptibility testing of the yeast Cryptococcus neoformans that shows promise for predicting the mycological response in patients to treatment. Quantitative cultures of the cerebrospinal fluid provide a direct measure of the patient's mycological response to treatment and have been used in multiple studies to identify the most promising antifungal drugs for subsequent testing in larger clinical studies. Using these quantitative measures of response, a modified macrobroth dilution assay system shows the potential for predicting the response of an individual patient to treatment with amphotericin B, fluconazole, or the combination of amphotericin B plus flucytosine. We describe this modified macrobroth dilution assay method, the statistical approach for assessing susceptibility, and the clinical decisions that can be guided by this in vitro antifungal drug susceptibility testing.     

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.     

Molecular typing and antifungal susceptibility of clinical sequential isolates of Cryptococcus neoformans from Sao Paulo State, Brazil

The antifungal susceptibility profiles and the genetic variability of 83 sequential clinical isolates of Cryptococcus neoformans, including four Cryptococcus gattii isolates, obtained from 38 Sao Paulo AIDS patients with cryptococcal meningitis were assessed by electrophoretic karyotyping and random amplified polymorphic DNA (RAPD) analysis. The majority of the Cryptococcus neoformans isolates were highly susceptible to amphotericin B and fluconazole. Twenty percent of the minimum inhibitory concentration values for amphotericin B varied from 0.5 to 1 micro g mL(-1). For fluconazole, 22% occurred in the range 8-16 mug mL(-1). Sequential isolates from nine patients showed a trend towards lower susceptibility to fluconazole, flucytosine, itraconazole and amphotericin B. The results of molecular typing by electrophoretic karyotyping and RAPD analysis showed the presence of 22 electrophoretic karyotypes (EK) and 15 RAPD profiles that were highly correlated. Our results provided evidence for the occurrence of genetic changes in some strains associated with microevolution during the course of infection. We also observed both microevolution and simultaneous coinfection with two distinct Cryptococcus neoformans strains in one patient. In some patients, we found changed EK- and RAPD patterns in association with increased MIC values.     

Two cation transporters Ena1 and Nha1 cooperatively modulate ion homeostasis, antifungal drug resistance, and virulence of Cryptococcus neoformans via the HOG pathway

Maintenance of cation homeostasis is essential for survival of all living organisms in their biological niches. It is also important for the survival of human pathogenic fungi in the host, where cation concentrations and pH will vary depending on different anatomical sites. However, the exact role of diverse cation transporters and ion channels in virulence of fungal pathogens remains elusive. In this study we functionally characterized ENA1 and NHA1, encoding a putative Na(+)/ATPase and Na(+)/H(+) antiporter, respectively, in Cryptococcus neoformans, a basidiomycete fungal pathogen which causes fatal meningoencephalitis. Expression of NHA1 and ENA1 is induced in response to salt and osmotic shock mainly in a Hog1-dependent manner. Phenotypic analysis of the ena1Δ, nha1Δ, and ena1Δnha1Δ mutants revealed that Ena1 controls cellular levels of toxic cations, such as Na(+) and Li(+) whereas both Ena1 and Nha1 are important for controlling less toxic K(+) ions. Under alkaline conditions, Ena1 was highly induced and required for growth in the presence of low levels of Na(+) or K(+) salt and Nha1 played a role in survival under K(+) stress. In contrast, Nha1, but not Ena1, was essential for survival at acidic conditions (pH 4.5) under high K(+) stress. In addition, Ena1 and Nha1 were required for maintenance of plasma membrane potential and stability, which appeared to modulate antifungal drug susceptibility. Perturbation of ENA1 and NHA1 enhanced capsule production and melanin synthesis. However, Nha1 was dispensable for virulence of C. neoformans although Ena1 was essential. In conclusion, Ena1 and Nha1 play redundant and discrete roles in cation homeostasis, pH regulation, membrane potential, and virulence in C. neoformans, suggesting that these transporters could be novel antifungal drug targets for treatment of cryptococcosis.     

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.     

Deletion of Cryptococcus neoformans AIF ortholog promotes chromosome aneuploidy and fluconazole-resistance in a metacaspase-independent manner

Apoptosis is a form of programmed cell death critical for development and homeostasis in multicellular organisms. Apoptosis-like cell death (ALCD) has been described in several fungi, including the opportunistic human pathogen Cryptococcus neoformans. In addition, capsular polysaccharides of C. neoformans are known to induce apoptosis in host immune cells, thereby contributing to its virulence. Our goals were to characterize the apoptotic signaling cascade in C. neoformans as well as its unique features compared to the host machinery to exploit the endogenous fungal apoptotic pathways as a novel antifungal strategy in the future. The dissection of apoptotic pathways revealed that apoptosis-inducing factor (Aif1) and metacaspases (Mca1 and Mca2) are independently required for ALCD in C. neoformans. We show that the apoptotic pathways are required for cell fusion and sporulation during mating, indicating that apoptosis may occur during sexual development. Previous studies showed that antifungal drugs induce ALCD in fungi and that C. neoformans adapts to high concentrations of the antifungal fluconazole (FLC) by acquisition of aneuploidy, especially duplication of chromosome 1 (Chr1). Disruption of aif1, but not the metacaspases, stimulates the emergence of aneuploid subpopulations with Chr1 disomy that are resistant to fluconazole (FLC(R)) in vitro and in vivo. FLC(R) isolates in the aif1 background are stable in the absence of the drug, while those in the wild-type background readily revert to FLC sensitivity. We propose that apoptosis orchestrated by Aif1 might eliminate aneuploid cells from the population and defects in this pathway contribute to the selection of aneuploid FLC(R) subpopulations during treatment. Aneuploid clinical isolates with disomies for chromosomes other than Chr1 exhibit reduced AIF1 expression, suggesting that inactivation of Aif1 might be a novel aneuploidy-tolerating mechanism in fungi that facilitates the selection of antifungal drug resistance.     

In vitro susceptibility characteristics of Cryptococcus neoformans varieties from AIDS patients in Goiânia, Brazil

Sixty clinical isolates of Cryptococcus neoformans from AIDS from Goiania, state of Goiás, Brazil, were characterized according to varieties, serotypes and tested for antifungal susceptibility. To differentiate the two varieties was used L-canavanine-glycine-bromothymol blue medium and to separate the serotypes was used slide agglutination test with Crypto Check Iatron. The Minimal Inhibitory Concentration (MIC) of fluconazole, itraconazole, and amphotericin B were determined by the National Committee for Clinical Laboratory Standards macrodilution method. Our results identified 56 isolates as C. neoformans var. neoformans serotype A and 4 isolates as C. neoformans var. gattii serotype B. MIC values for C. neoformans var. gattii were higher than C. neoformans var. neoformans. We verified that none isolate was resistant to itraconazole and to amphotericin B, but one C. neoformans var. neoformans and three C. neoformans var. gattii isolates were resistant to fluconazole. The presence of C. neoformans var. gattii fluconazole resistant indicates the importance of determining not only the variety of C. neoformans infecting the patients but also measuring the MIC of the isolate in order to properly orient treatment.     

Comparative Analysis of the Vitek 2 Antifungal Susceptibility System and E-test with the CLSI M27-A3 Broth Microdilution Method for Susceptibility Testing of Indian Clinical Isolates of Cryptococcus neoformans

The emergence of antifungal resistance among Cryptococcus neoformans isolates is a matter of great concern. The Clinical and Laboratory Standards Institute (CLSI) broth microdilution reference method (BMD) for antifungal susceptibility testing of C. neoformans is tedious and time-consuming. Consequently, there is a greater need for a reproducible in vitro susceptibility testing method for use in clinical microbiology laboratories. By random amplified polymorphic DNA analysis, the 62 Indian clinical isolates were characterized as Cryptococcus neoformans var. grubii. We evaluated the susceptibilities of these isolates for amphotericin B (AMB) and fluconazole (FLC) by two commercial techniques, i.e., Vitek 2 and E-test against the CLSI M27-A3 BMD. The essential agreement (EA) between the Vitek 2 and E-test with the reference procedure for FLC was similar (82.2%). For AMB, EA of 92 and 76% was obtained with E-test and Vitek 2. Excellent categorical agreement (CA) (98.3% and 100% by Vitek 2 and E-test, respectively) was obtained for AMB. The CA for FLC was 81 and 77.4% by Vitek 2 and E-test. We conclude that both E-test and Vitek 2 system have acceptable levels of accuracy for susceptibility testing of both the drugs. Both of them could identify fluconazole-resistant strains. Vitek 2 could be used for testing susceptibility of voriconazole and 5-flucytosine also at the same time.     

以反复多发脓肿为表现的播散型隐球菌病1例

报道以反复多发脓肿为表现的播散型隐球菌病患者1例。患者男,68岁,因＂全身反复多发性脓肿1a,伴低热2个月余＂入院,该患者以背部脓肿起病,反复发作累及肺部、骨骼、皮肤等多部位,多次组织及血标本病原学检查、隐球菌乳胶凝集试验均阴性,给予多种抗菌药物治疗无效,后在右髂部脓肿切开组织物中培养到新生隐球菌。经脓肿切开引流及静脉滴注两性霉素B脂质体联合伊曲康唑治疗后,脓肿消退,症状消失,改用氟康唑联合氟胞嘧啶治疗4个月后停药,并已随访至今7个月,患者未有发热及新的脓肿出现。     

Combinatorial approach to lead optimization of a novel hexapeptide with antifungal activity

Three sets of sublibraries of an antifungal lead peptide His-D-Trp-D-Phe-Phe-D-Phe-Lys-NH2 (I) have been prepared by introducing variations at positions 1, 4 and 6. They were screened for their antifungal activity against C. albicans and C. neoformans in order to quantify inhibition at each step of the hexapeptide sublibrary iteration. The studies led to the identification of Arg-D-Trp-D-Phe-Ile-D-Phe-His-NH2 as a novel hexapeptide with potent antifungal activity against both C. albicans and C. neoformans.     

The Cryptococcus neoformans MAP kinase Mpk1 regulates cell integrity in response to antifungal drugs and loss of calcineurin function

Cell wall integrity is crucial for fungal growth, development and stress survival. In the model yeast Saccharomyces cerevisiae, the cell integrity Mpk1/Slt2 MAP kinase and calcineurin pathways monitor cell wall integrity and promote cell wall remodelling under stress conditions. We have identified the Cryptococcus neoformans homologue of the S. cerevisiae Mpk1/Slt2 MAP kinase and have characterized its role in the maintenance of cell integrity in response to elevated growth temperature and in the presence of cell wall synthesis inhibitors. C. neoformans Mpk1 is required for growth at 37 degrees C in vitro, and this growth defect is suppressed by osmotic stabilization. C. neoformans mutants lacking Mpk1 are attenuated for virulence in the mouse model of cryptococcosis. Phosphorylation of Mpk1 is induced in response to perturbations of cell wall biosynthesis by the antifungal drugs nikkomycin Z (a chitin synthase inhibitor), caspofungin (a beta-1,3-glucan synthase inhibitor), or FK506 (a calcineurin inhibitor), and mutants lacking Mpk1 display enhanced sensitivity to nikkomycin Z and caspofungin. Lastly, we show that calcineurin and Mpk1 play complementing roles in regulating cell integrity in C. neoformans. Our studies demonstrate that pharmacological inhibition of the cell integrity pathway would enhance the activity of antifungal drugs that target the cell wall.     

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.     

Capsule enlargement in Cryptococcus neoformans confers resistance to oxidative stress suggesting a mechanism for intracellular survival

Cryptococcus neoformans is a facultative intracellular pathogen. The most distinctive feature of C. neoformans is a polysaccharide capsule that enlarges depending on environmental stimuli. The mechanism by which C. neoformans avoids killing during phagocytosis is unknown. We hypothesized that capsule growth conferred resistance to microbicidal molecules produced by the host during infection, particularly during phagocytosis. We observed that capsule enlargement conferred resistance to reactive oxygen species produced by H(2)O(2) that was not associated with a higher catalase activity, suggesting a new function for the capsule as a scavenger of reactive oxidative intermediates. Soluble capsular polysaccharide protected C. neoformans and Saccharomyces cerevisiae from killing by H(2)O(2). Acapsular mutants had higher susceptibility to free radicals. Capsular polysaccharide acted as an antioxidant in the nitroblue tetrazolium (NBT) reduction coupled to beta-nicotinamide adenine dinucleotide (NADH)/phenazine methosulfate (PMS) assay. Capsule enlargement conferred resistance to antimicrobial peptides and the antifungal drug Amphotericin B. Interestingly, the capsule had no effect on susceptibility to azoles and increased susceptibility to fluconazole. Capsule enlargement reduced phagocytosis by environmental predators, although we also noticed that in this system, starvation of C. neoformans cells produced resistance to phagocytosis. Our results suggest that capsular enlargement is a mechanism that enhances C. neoformans survival when ingested by phagocytic cells.