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阴道念珠菌菌种鉴定及其耐药性分析

【摘要】    目的 回顾2003~2006年,海南省人民医院妇科门诊念珠菌阴道炎的473株念珠菌的鉴定结果及药敏特点,为临床用药治疗提供参考。方法 按常规霉菌培养分离菌株采用生物梅里埃Vitek-AMS-32及YBC鉴定卡全自动检测鉴定,药敏采用生物梅里埃ATB-FUNGUSTS试条,对5-氟胞嘧啶、两性霉素B、酮康唑、制霉菌素、益康唑、咪康唑等6种药敏进行测定。结果 检出473株念球菌中,共鉴定出8种念珠菌,其中白色念珠菌346株占73.1%,其次为光滑球拟酵母45株占9.5%,热带念珠菌44株占9.3%,季也蒙念球菌13株占2.7%,此外还有占份量较少的4种。药敏结果制霉菌素和两性霉素B对念珠菌的抑菌作用较强,耐药率较低,分别为5.3%和8.2%,对其余耐药率从低到高依次为酮康唑28.1%,益康唑38.1%较大的差异。结论 白色念珠菌仍是妇科霉菌性阴道炎的主要病原菌,但其它念珠菌种构成比的增加应引起我们足够的重视,加强念珠菌药敏的测试,才能做到合理用药、科学治疗。

【关键词】  念珠菌;阴道炎;耐药率

  Identification of candid species in vaginitis patients and analysis of drug resistance.
 
  WANG Xu-ming, MO Cheng-jin.

  Hainan Provincial People’s Hospital, Haikou 570311, Hainan, P.R.China
   
  Abstract:Objective  To summarize the results of identification of 473 Candida strains and their sensitivity to antibiotics.  Methods  The 473 candida strains were isolated  by Bimreieux Vitek-AMS-32 and identified by YBC card. The sensitivity of the 473 Candida strains to six antibiotics were determined by Bimreieux ATB-FUNGUSTS.  Results  There 8 species of Candida are detected in the 473 strains. The number of Candida albicans, Torulopsis glabrata, Candida tropicalis and Candida Guilliermondii were 346 (73.2%), 45 (9.5%),44 (9.3%) and 13 (2.7%) respectively. There were also other 4 species. The resistance rates of Candida to 5-fluorocytosine, amphotericin B, ketoconazole, anticandine, econazole and miconazole were 42.5%, 8.2%, 28.1%, 5.3%, 38.1% and 59.4% respectively. The same drug has different effect on different species of Candida.  Conclusion  Candida albicans is still the major pathogenic bacterium although other species increased. The test of sensitivity of Candida to antibiotics is useful for guiding clinical treatmetn.
   
  Key words:Candida; Vaginitis; Drug resistance rate

    念珠菌(现称假绿酵母菌)阴道炎是妇科最常见的疾病之一,近年来有不断增加趋势。特别是临床抗真菌药物的广泛应用以及患者自己购药的不规范治疗,以致造成感染菌株变迁和耐药菌株的不断增加,使该病难以治愈,常有反复。为了给临床治疗提供参考,现将海南省人民医院部分霉菌性阴道炎的念珠菌鉴定结果及药敏总结如下。

  1  材料与方法

  1.1  标本来源 

  均来自海南省人民医院2003~2006年妇科门诊送检的阴道分泌物,患者年龄最小16岁,最大54岁,平均年龄39岁。

  1.2 细菌培养与鉴定 

  标本接种沙保罗氏平皿及血平皿,置35℃恒温普通培养箱培养1~7d。菌株鉴定采用生物梅里埃Vitek-AMS-32全自动微生物分析仪以及与之配套的念珠鉴定卡YBC。

  1.3 药敏检测 

  采用生物梅里埃ATB-FUNGUSTS试条,实验操作按说明书进行。

  1.4 质控菌株 

  白色念珠菌ATCC 14053。

  2 结果

    检出的473株霉菌鉴定结果:白色念珠菌346株占73.1%,其余菌株占构成比详见表1。药敏结果显示,念球菌对6种抗真菌药物的耐药率由低到高依次为制霉菌素5.3%,两性霉素B 8.2%,酮康唑28.1%,益康唑38.1%,5-氟胞嘧啶42.5%,咪康唑59.4%。而不同菌种对同一种药物的敏感性存在有较大的差异,详见表2。表1  检出73株念珠菌的菌种占构成比菌种名称检出株数占构成比(略)表2  473株念珠菌对6种抗真菌药的的耐药率(略)注:合计行的耐药率为耐药合计株数/473,“( )”内是耐药菌株数。

  3 讨论

  3.1 对检出473株霉菌的鉴定结果,共鉴定为8种念珠菌,其中白色念珠菌占73.1%,比国内朱慧兰[1]2001年报告的79.3%略低。霉菌性阴道炎仍是白色念珠菌为主要致病菌,但其它念珠菌的构成比在增加,品种也多样化,这种变化的趋势,与其它临床体内真菌感染变迁基本符合。据张军民等2007年报告[2],近年来白色念珠菌占的构成比在下降,而光滑念珠菌和近平滑念珠的构成比在显著增加,且新的菌种也在增多。其原因可能与临床对白色念珠菌的有效治疗,使其它念珠菌生长旺盛,以填补生态学小环境平衡有关,因此对非白色念珠引起的阴道炎,也应引起我们足够的重视。

  3.2 对473株阴道念珠菌的药敏测试结果中,阴道念珠菌对制霉菌素和两性霉素B的耐药率最低,分别为5.3%和8.2%,与顾志群[3]2006年报告相近,因此可做为临床治疗念珠菌阴道炎的首选药物。但是与临床上非阴道念珠菌药敏相比较,阴道念珠菌的耐药率有增高的趋势。表2中两性霉素B的耐药率为8.2%,酮康唑为28.1%,5-氟胞嘧啶为42.5%,明显高于章强强2000年的报告[4]。另外,表2中白色念球菌对两性霉素B和酮康唑的敏感率可计算出分别是93.1%和74.0%,也低于张军民2007年报告[2]的100%和93.8%。念珠菌阴道炎治疗复发率较高,患者多反复体外用药治疗或自己购买药物的不规范治疗,可能是造成耐药菌株增多的主要原因。

  3.3 从表2中可看出不同菌种对同一种药物的耐药性存在有较大的差异,如光滑球拟酵母菌对制霉菌素的耐药率(11.1%)高于白色念珠菌(5.2%)和热带念珠菌(4.5%),光滑球拟酵母菌对两性霉素B的耐药率(20.0%)也明显高于热带念珠菌(6.8%),类似情况还有酮康唑、5-氟胞嘧啶、咪康唑等。阴道念珠菌不同菌种间这种耐药性明显差异的特点,要求我们在实际工作中必须加强念珠菌的培养和鉴定以及药敏测试的工作,只有这样才能做到合理用药、有效治疗,避免盲目用药,造成耐药菌株增多。

【参考文献】
    [1]朱慧兰, 谷进, 李平,等. 外阴阴道念珠菌病的病原检测及体外药敏检测[J]. 中国抗感染化疗杂志, 2001, 3: 175~176.

  [2]张军民, 席丽艳, 鲁长明,等. 医院分离深部真菌菌株流行病学调查和药敏试验研究[J]. 中华检验医学杂志, 2007, 30: 27~29.

  [3]顾志群. 383例阴道念珠菌感染群鉴定及药敏分析[J]. 滨州医学院报, 2006, 29(6): 432~433.

  [4]章强强, 苏逸丹, 李莉,等. 100株临床分离致病酵母体外药敏试验分析[J]. 中华皮肤科杂志, 2000, 33: 5.


作者单位:海南省人民医院检验科,海南 海口 570102.

日期:2010年1月13日 - 来自[2008年第8卷第2期]栏目
循环ads

Coculture of THP-1 Human Mononuclear Cells with Candida albicans Results in Pronounced Changes in Host Gene Expression

    Departments of Pharmacy and Pharmaceutical Sciences, College of Pharmacy
    Department of Pediatrics, College of Medicine, University of Tennessee Health Science Center
    Children's Foundation Research Center, Le Bonheur Children's Medical Center, Memphis, Tennessee

    Background.

    The host's first line of defense against bloodstream infection with Candida albicans involves the recognition and clearance of the fungus by neutrophils and monocytes/macrophages. The purpose of the present study was to examine changes in the monocytic cell gene-expression profile in response to C. albicans stimulation.

    Methods.

    RNA was isolated from THP-1 cells 3 h after coculture with live C. albicans SC5314 cells. After hybridization to microarrays, genes differentially expressed by at least 2.0-fold were included in the final data set.

    Results.

    As expected, TNFA, IL8, CD83, MIP1A, and MIP1B were among the genes up-regulated. This was confirmed by real-time reverse-transcriptase polymerase chain reaction (RT-PCR), fluorescence-activated cell sorting analysis, and enzyme-linked immunosorbent assay. Furthermore, RGS1, RGS2, RGS16, DSCR1, GROB, EGR3, FLT4, and TNFAIP6 were also up-regulated in response to C. albicans, whereas CCR2 and NCF2 were among the genes down-regulated in response to C. albicans. Differential expression of selected genes was confirmed at several time points by real-time RT-PCR.

    Conclusions.

    This study defines the gene expression profile of an early response of human mononuclear cells to C. albicans and identifies genes not previously known to be responsive to this pathogen.

    The human opportunistic pathogen Candida albicans causes superficial and disseminated disease in immunocompromised individuals. Superficial C. albicans infections occur most often in the oropharynx and vagina. Although not invasive or life threatening, oropharyngeal candidiasis is one of the most common infections in persons with HIV/AIDS. In contrast, disseminated candidiasis is deadly, accounting for the highest incidence of mortality (40%) of any cause of bloodstream infections [1], and it remains one of the leading causes of death in neutropenic patients with cancer [2].

    Competent host response to disseminated candidiasis involves neutrophils and mononuclear phagocytes for recognition and clearing of fungal cells [3]. In addition to their role as phagocytic cells, both mononuclear phagocytes and neutrophils are capable of secreting immunomodulatory cytokines that influence the host immune response to fungal infection [4, 5]. C. albicansstimulated monocytes, as well as stimulated CD4+ and CD8+ T cells and NK cells, produce macrophage inflammatory protein (MIP)1, MIP-1, and RANTES, which are responsible for chemoattraction of activated CD4+ Th1 T cells, dendritic cells (DCs), and monocytes to the site of infection [6]. Additionally, monocytes produce interleukin (IL)1, tumor necrosis factor (TNF), and IL-10 in response to C. albicans hyphae and produce IL-12 in response to C. albicans unable to form hyphae [79].

    C. albicans interacts with monocytes through Toll-like receptors (TLRs) 2 and 4 [10], the integrin CD11b/CD18 [11], and the  glucan receptor dectin-1 [12]. Intracellularly, signaling involves at least mitogen-activated protein kinase and protein kinase C pathways to induce expression of host factors [13]. It remains unresolved whether other pathways, such as extracellular-related kinases, are also involved in the production of factors such as chemokines in response to C. albicans.

    The human monocytic cell line THP-1 affords a competent in vitro model of monocytes/macrophages during interaction with fungal cells. Previous studies have utilized THP-1 cells to examine human monocyte/macrophage chemokine production in response to whole fungal cells or fungal cell wall components [14, 15], phagocytosis of fungal cells [16], and differentiation and cell surface marker expression [17, 18]. THP-1 cells have proven advantageous in microarray analyses, since, in addition to their established usefulness as a monocyte/macrophage model, their homogeneous genetic background minimizes the amount of variability in the resulting gene expression profiles [19, 20]. Therefore, because of their established function as a model of peripheral blood mononuclear cells (PBMCs) and their attractiveness for use in microarray analysis, we chose to use THP-1 cells in an in vitro model of host monocyteC. albicans interaction.

    In the present study, to better explore the impact of C. albicans on host monocyte gene expression, we simultaneously examined the expression of 18,400 human genes by use of microarray hybridization of RNA from THP-1 cells cocultured for 3 h with C. albicans strain SC5314. Further consideration was given to several genes with known involvement in the host response to C. albicans, by examining mRNA and protein expression during a span of 12 h in THP-1 cells cocultured with this fungus. In addition, several genes whose expression has never before been associated with the host response to C. albicans were examined during the time course by real-time reverse-transcriptase polymerase chain reaction (RT-PCR), to identify differential mRNA expression.

    MATERIALS AND METHODS

    Human cell line and C. albicans isolate.

    The THP-1 human monocytic cell line (American Type Culture Collection) was used in this study. Cells were maintained in culture medium (RPMI 1640 and 10% fetal calf serum) at 37°C in a humidified chamber containing 5% CO2. SC5314 is a wild-type, virulent strain capable of producing hyphae. It was stored as a glycerol stock at -70°C and was grown in yeast nitrogen base broth containing 5% dextrose at 30°C in a shaking incubator.

    Coculture conditions.

    Overnight fungal cultures were washed, resuspended in culture medium, and incubated in a shaking incubator for 3 h. THP-1 cells were also washed, counted using a hemacytometer, plated at 2 × 106 cells/well, and allowed to equilibrate at 37°C for 3 h. After incubation, fungal cultures were washed, counted using a hemacytometer, and plated with THP-1 cells at a fungus-monocyte ratio of 3 : 10. This ratio was determined (data not shown) to preserve cell viability while providing suitable host gene response to known response genes, such as TNFA. Cocultures were incubated at 37°C in a CO2 incubator for 3 h (for microarray hybridization) or for 112 h (for subsequent analyses). After incubation, each coculture was examined by light microscopy; the majority of C. albicans cells had formed hyphae by 1 h, and many C. albicans cells were intracellular by 6 h. Viability of THP-1 cells was assessed by trypan blue exclusion (80% viability was observed), supernatants were collected, and RNA was isolated from THP-1 cells. Supernatants from cocultures were tested using an E-TOXATE kit (Sigma Chemical) and contained <0.06 EU/mL endotoxin. All experiments were performed in duplicate.

    Total RNA isolation.

    Total RNA was isolated using Trizol reagent (Gibco/Invitrogen) in accordance with the manufacturer's instructions. RNA pellets were suspended in diethylpyrocarbonate-treated water and stored at -70°C. The integrity of RNA samples was assessed using an Agilent Bioanalyzer before microarray hybridization and by gel electrophoresis before real-time RT-PCR analysis.

    Microarray hybridization and data analysis.

    Differential gene expression was measured by hybridizing Affymetrix U133A arrays and comparing normalized signals between THP-1 cells cultured in medium alone and those cultured with C. albicans. Two sets of hybridizations were performed using RNA samples generated from 2 independent coculture experiments. Ten micrograms of total RNA was subjected to first- and second-strand cRNA synthesis incorporating biotin-labeled nucleotides. cRNA was fragmented and subsequently hybridized overnight with microarray chips, using the manufacturer's hybridization buffer. Hybridized microarrays were washed and subjected to a signal-enhancement protocol consisting of an initial incubation with streptavidinphycoerythrin (PE) conjugate, followed by staining with goat anti-streptavidin biotinylated antibody and a final staining with the streptavidin-PE conjugate. The microarrays were scanned using the GeneArray scanner with an argon ion laser excitation source, and emission was detected by a photomultiplier tube through a 570-nm long-pass filter. Digitized image data were processed using GeneChip Operating Software (Affymetrix). Data normalization was performed as described elsewhere [21]. Genes were considered to be up-regulated if averaged normalized ratios were 2.0 and were considered to be down-regulated if averaged normalized ratios were -2.0.

    cDNA synthesis and real-time RT-PCR.

    First-strand cDNAs were synthesized from 2 g of total RNA in a 21-L reaction volume by use of the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). Quantitative real-time PCRs were performed in duplicate using the 7000 Sequence Detection System (Applied Biosystems). Independent PCRs were performed in triplicate, using the same cDNA for both the gene of interest and 18S rRNA, by use of the SYBR Green PCR Master Mix (Applied Biosystems). Gene-specific primers were designed for the gene of interest and 18S rRNA by use of Primer Express software (version 2.0; Applied Biosystems) and the Oligo Analysis & Plotting Tool (Qiagen) and are listed in table 1. The PCR conditions consisted of AmpliTaq Gold activation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min. To verify that a single product was amplified, a dissociation curve was generated at the end of each PCR cycle, by use of software provided with the 7000 Sequence Detection System. The change in fluorescence of SYBR Green I dye in every cycle was monitored by the system software, and the cycle threshold (CT) above background for each reaction was calculated. The CT value of 18S rRNA was subtracted from that of the gene of interest to obtain a CT value. The CT value of the least abundant sample at all time points for each gene was subtracted from the CT value of each sample to obtain a CT value. The gene expression level relative to the calibrator was expressed as 2-CT [22].

    ELISAs.

    MIP-1, MIP-1, IL-8, and TNF- concentrations were determined by use of commercial ELISA kits (R&D Systems). Supernatants were stored at -70°C until assayed. Experiments yielding supernatants were performed independently in duplicate, and each supernatant was plated in duplicate in the ELISA. Optical densities were read at the appropriate wavelength on a microplate reader, and measurements were calculated as means ± SEs.

    Fluorescence-activated cell sorting (FACS) analysis.

    C. albicansTHP-1 cell cocultures were performed as described above, except that the coculture incubation time was 6 h. Each culture was split into two 5-mL round-bottom tubes, and cells were collected briefly by centrifugation and washed twice in PBS. Cells were incubated with 20 L of either an antihuman CD83 monoclonal antibody or isotype control (both labeled with PE from Pharmingen) at 4°C for 30 min, washed twice in PBS, and resuspended in 0.5 mL of 1% paraformaldehyde. All samples were kept on ice until analyzed. Cell surface expression of CD83 was assessed on a Becton Dickinson FACSCalibur flow cytometer, with >1 × 104 events collected for each sample. Cells were gated according to light-scatter properties to exclude cellular debris. Gating for fluorescence intensity was determined by manually gating in the isotype control medium-cultured THP-1 cell sample and maintaining that gating for subsequent samples. Two replicate experiments were performed.

    RESULTS

    The comparison of the gene-expression profiles of C. albicansstimulated and unstimulated THP-1 cells revealed 131 genes differentially expressed by at least 2.0-fold (table 2). Of these, 47 genes were up-regulated, and 84 genes were down-regulated. The up-regulated antipathogen-response genes included MIP1B, MIP1A, and TNFA. Signal-transduction genes found to be up-regulated included DSCR1 (Down syndrome critical region 1), EGR3 (early growth response 3), RGS1 (regulator of G protein signaling), and FLT4 (fms-related tyrosine kinase 4). Pol II transcription genes that were down-regulated in THP-1 cells in response to C. albicans stimulation included LMYC and CEBPA (CCAAT/enhancer binding protein ). Other genes of interest that were down-regulated in THP-1 cells in response to C. albicans stimulation were the IL-10 receptor antagonist IL10RA and the chemokine receptor CCR2.

    Further analysis was performed on several antipathogen-response genes and their gene products that are known to be responsive to C. albicans, by following their expression over time in response to C. albicans stimulation. Real-time RT-PCR revealed early, maximal expression (by 3 h) of TNFA, MIP1A, CD83, and MIP1B mRNA in THP-1 cells cocultured with C. albicans (figure 1). In these same cells, IL8 mRNA expression reached maximal expression levels by 9 h.

    Supernatants from THP-1 cells cocultured with C. albicans or in medium alone were used to measure cytokine/chemokine levels by ELISA (figure 2). As expected, cells stimulated by C. albicans produced significantly more IL-8, TNF-, MIP-1, and MIP-1 protein than did cells cultured in medium alone. CD83 protein expression at 6 h was assessed by surface staining of stimulated and unstimulated THP-1 cells with PE-labeled antihuman CD83 monoclonal antibody and subsequent analysis by flow cytometry (figure 3). Although there was modest surface expression of CD83 on cells cultured in medium alone, there was an increase in the level of surface expression of CD83 on cells stimulated with C. albicans. Specifically, the mean channel on the FACS histogram shifted from 7.55 for medium-stimulated cells to 16.37 for C. albicansstimulated cells, suggesting that each THP-1 cell analyzed by FACS increased the number of CD83 molecules on its surface. Surprisingly, IL1B failed to reach the minimum cutoff of 2-fold difference in expression in the microarray analysis. IL1B was therefore examined by real-time RT-PCR over time. IL1B, like IL8, also reached its maximal level of expression by 9 h (figure 4).

    Several genes previously not known to be involved in the host response to C. albicans were also selected for further examination of mRNA expression by real-time RT-PCR (figure 4). These included DSCR1, RGS1, RGS2, RGS16, GROB, EGR3, FLT4, CCR2, TNFAIP6, and NCF2. DSCR1, RGS2, GROB, and FLT4 had expression patterns similar to those of TNFA, IL1B, MIP1A, and MIP1B. RGS1 exhibited an expression pattern similar to that of IL8, with a maximal expression of nearly 40-fold at the 6-h time point that was sustained for the remainder of the time course. NCF2 and CCR2 exhibited an expression pattern that was inverse to that of RGS1 and IL8, with expression levels decreasing at least 2-fold by the 6-h time point. RGS16 and TNFAIP6 exhibited later maximal expression similar to that of RGS1 and IL8, but their expression levels decreased at later time points.

    DISCUSSION

    Induction by C. albicans of expression of antipathogen response genes in THP-1 cells.

    Among the most highly represented up-regulated genes were those involved in the antipathogen response, with MIP1A and MIP1B the most up-regulated genes identified by microarray. Interestingly, IL8 mRNA production was much greater at later time points than at 3 h, when RNA was harvested for microarray hybridization, suggesting that IL8 may respond to factors produced earlier in the stimulation. Although it did not make the 2-fold cutoff for inclusion in the list of differentially expressed genes, with an average of 1.8-fold expression (data not shown), IL1B was examined by real-time RT-PCR time course analysis, since its expression in human leukocytes was previously associated with response to C. albicans infection [9]. The analysis indicated that IL1B levels were at least 2-fold higher at every time point in C. albicansstimulated cells than in medium-cultured cells.

    Some C. albicansspecific, antipathogen-response genes we did not see in our list of differentially expressed genes were IL10, IL12A, IL12B, and SCYA5 (RANTES). IL10 has been shown to be up-regulated in monocytes in response to filamentous C. albicans. IL12A and IL12B, genes that encode the p35 and p40 subunits of IL-12 p70, have been demonstrated to be up-regulated in response to yeast forms of C. albicans. RANTES has also been shown to be expressed in response to C. albicans stimulation. Because the experiments in the present study were performed with filamenting C. albicans, it was not surprising to not see up-regulation of IL12A or IL12B. One study reported detection of IL10 mRNA in DCs in response to hyphae at 18 h after stimulation [23]. RANTES mRNA expression is reported in the literature to be increased slightly at 3 h and greatly increased at 20 h after stimulation in PBMCs; however, the expression level is not quantified from the Northern hybridizations shown [6].

    TNFA was also up-regulated in C. albicansstimulated THP-1 cells. Several studies have described the increased expression of this cytokine in monocyte, granulocyte, or PBMC cultures with C. albicans [2427]. Additionally, we observed that TNFA mRNA induction is at its highest level within 1 h of coculture and is virtually at its maximal protein level by 3 h. Such an early TNFA response may be critical and responsible for the induction of many of the other molecules in the gene list. For example, ATF3 (activating transcription factor 3), DSCR1, and RGS16 are inducible by TNF- [2830]. TNF- also strengthens the function of monocyte-derived CD83+ DCs by enhancing their proliferation in the presence of C. albicans, protecting their phagocytic ability, and enhancing their allogeneic T-cell stimulatory activity [31].

    The up-regulation of CD83 in THP-1 cells was somewhat surprising, since it is a marker on mature DCs. However, monocytes stimulated with C. albicans hyphae had increased expression of CD83, although they possessed characteristics atypical of DCs [25]. Although CD83 is primarily used as a cell surface determinant, studies designed to determine a potential role of the molecule in DCs have shown that soluble forms to be involved in modulating the immune response of T cells by inhibiting DC-driven allogeneic and peptide-specific T cell proliferation while inhibiting the maturation of DCs by causing the down-regulation of CD80 and CD83 on immature DCs [32].

    TNFAIP6 (also known as TNF-stimulated gene 6, or TSG6) is expressed in mononuclear cells, among other cell types, in response to TNF- and IL-1. It is thought to function as an anti-inflammatory molecule, as part of a negative feedback loop during inflammation [33]. It also acts to inhibit protease action during inflammation, by forming stable complexes with components of the serine protease inhibitor inter- inhibitor (II), which inhibits the protease activity of plasmin, important in the protease network associated with inflammation. The induction of TNFAIP6 is consistent with the expression of TNFA in response to C. albicans stimulation.

    CCR2, which is down-regulated 2.4-fold in response to C. albicans, is a G proteincoupled receptor for the chemokines monocyte chemotactic protein (MCP)1, MCP-3, and MCP-4. Examination of a pulmonary Cryptococcus neoformans infection model in CCR2 knockout mice revealed that these mice had a prolonged duration of disease and were less able to recruit macrophages and CD8+ T cells into the lung [34]. These mice were found to have a Th2-type response, chronic pulmonary eosinophilia, and high serum IgE levels, suggesting that CCR2 is required for the development of a Th1 response to C. neoformans. Additionally, studies of the maturation of DCs revealed that expression of CCR2 mRNA was down-regulated to nondetectable levels [35].

    The protein encoded by NCF2 (neutrophil cytosol factor 2, or p67phox) is the limiting cofactor in the assembly of the NADPH oxidase enzyme complex in neutrophils. NADPH oxidase catalyzes the production of oxygen radicals that are essential in the defense against pathogens, and the NCF2 gene product is involved in the final activation of the enzyme complex. Although TNF-treated monocytic cells have NCF2 up-regulation [36], the present study indicates that NCF2 is down-regulated in the presence of increased levels of TNF-. It is possible that some other factor produced in response to C. albicans is overriding the effect of TNF- in modulating the expression of NCF2 in C. albicansstimulated cells.

    GROB (or MIP2A), up-regulated >3-fold in this study, is produced by activated monocytes and neutrophils at the site of inflammation. It enhances neutrophil function by increasing CD11b cell surface expression, superoxide production, chemotaxis, and enhancing killing [37]. GROB also enhances superoxide production in monocytes and has recently been shown to be produced by monocyte-derived DCs in response to bacterial flagellar proteins or lipopolysaccharide [38].

    Differential expression of signal transduction molecules in C. albicansstimulated THP-1 cells.

    DSCR1 is a gene found in the chromosome 21 Down syndrome critical region. Recently, it was found to be involved in putative negative feedback regulation after vascular endothelial growth factor (VEGF) stimulation in endothelial cells [29]. Similar to cyclosporin A, it is antagonistic to calcineurin signaling, resulting in down-regulation of several VEGF-responsive genes, such as ESEL (E-selectin). These genes have been shown to be up-regulated in endothelial cells upon stimulation with C. albicans [39]. It is possible that DSCR1 up-regulation in C. albicansstimulated THP-1 cells is involved with the normal negative regulation of monocyte-specific molecules during the inflammatory process.

    The molecules RGS1, RGS2, and RGS16 were up-regulated 4-fold in response to SC5314. Each are involved with regulating GTPase activity of the G subunit of G proteincoupled receptors, diminishing the duration of downstream signaling that occurs. Previously, it was shown that disruption of RGS1 in mice leads to abnormal trafficking of antibody-secreting cells, as well as to abnormalities in the spleen and Peyer patches [40]. Another study demonstrated that TLR signaling in human monocyte-derived DCs leads to increased RGS1 and RGS16 expression [41]. In short, it seems that the RGS gene products help to ensure normal responses of monocyte-derived DCs through TLRs and chemokine receptors.

    EGR3 is a zinc-finger transcription factor and an immediate-early gene product. It was up-regulated nearly 5-fold in response to C. albicans. Expression of EGR3 is inhibited by cyclosporin A and can be induced by a variety of external stimuli [42]. EGR3 activates transcription of many genes, including FasL, TRAIL, and TNFA [43].

    FLT4, which is up-regulated >2-fold in response to C. albicans, is a VEGF receptor typically found on the surface of endothelial cells. A recent study reports FLT4 protein expression on the surface of immature DCs that were derived from CD14+ monocytes cultured with granulocyte-macrophage colony-stimulating factor and IL-4 [44]. These immature DCs also expressed CD1a, HLA-DR, and CD86, as well as endothelial cell markers such as VE-cadherin and FLT1. However, as these cells were allowed to mature in the presence of TNF-, they lost their expression of endothelial cell markers in favor of CD83 expression. The role of FLT4 in THP-1 cells in response to C. albicans expression is unclear.

    Down-regulation of protein-synthesis genes in response to C. albicans stimulation.

    Four protein-synthesis genes were down-regulated in cells cocultured with C. albicans. Since there are >30 genes involved in translation initiation, the down-regulation of these genes was probably not indicative of down-regulation of protein synthesis in general. In fact, one of these genes, eIF5A, has been demonstrated recently to be a regulator of p53 [45]. Up-regulation of eIF5A leads to p53 up-regulation and increased probability of apoptosis. Therefore, down-regulation of eIF5A in the present study may have contributed to the proliferation of THP-1 cells in response to C. albicans stimulation.

    Conclusions.

    The present study provides important information about the gene-expression profile of human monocyte-like cells in response to C. albicans. Identification of newly identified genes provides insight into the regulation of the antipathogen response, while time course studies indicate the dynamics of the response. Future studies examining the role of the RGS genes, DSCR1, EGR3, and FLT4 in the host response to C. albicans, especially pertaining to their interaction with TLRs or other C. albicansinteracting molecules, is warranted.

    References

    1.  Edmond MB, Wallace SE, McClish DK, Pfaller MA, Jones RN, Wenzel RP. Nosocomial bloodstream infections in United States hospitals: a three-year analysis. Clin Infect Dis 1999; 29:23944. First citation in article

    2.  Viscoli C, Girmenia C, Marinus A, et al. Candidemia in cancer patients: a prospective, multicenter surveillance study by the Invasive Fungal Infection Group (IFIG) of the European Organization for Research and Treatment of Cancer (EORTC). Clin Infect Dis 1999; 28:10719. First citation in article

    3.  Diamond RD, Oppenheim F, Nakagawa Y, Krzesicki R, Haudenschild CC. Properties of a product of Candida albicans hyphae and pseudohyphae that inhibits contact between the fungi and human neutrophils in vitro. J Immunol 1980; 125:2797804. First citation in article

    4.  Cassatella MA, Meda L, Gasperini S, D'Andrea A, Ma X, Trinchieri G. Interleukin-12 production by human polymorphonuclear leukocytes. Eur J Immunol 1995; 25:15. First citation in article

    5.  Lloyd AR, Oppenheim JJ. Poly's lament: the neglected role of the polymorphonuclear neutrophil in the afferent limb of the immune response. Immunol Today 1992; 13:16972. First citation in article

    6.  Huang C, Levitz SM. Stimulation of macrophage inflammatory protein1, macrophage inflammatory protein1, and RANTES by Candida albicans and Cryptococcus neoformans in peripheral blood mononuclear cells from persons with and without human immunodeficiency virus infection. J Infect Dis 2000; 181:7914. First citation in article

    7.  Aybay C, Imir T. Tumor necrosis factor (TNF) induction from monocyte/macrophages by Candida species. Immunobiology 1996; 196:36374. First citation in article

    8.  Trinchieri G. The two faces of interleukin-12: a pro-inflammatory cytokine and a key immunoregulatory molecule produced by antigen-presenting cells. Ciba Found Symp 1995; 195:20314. First citation in article

    9.  Xiong J, Kang K, Liu L, Yoshida Y, Cooper KD, Ghannoum MA. Candida albicans and Candida krusei differentially induce human blood mononuclear cell interleukin-12 and gamma interferon production. Infect Immun 2000; 68:24649. First citation in article

    10.  Netea MG, Van Der Graaf CA, Vonk AG, Verschueren I, Van Der Meer JW, Kullberg BJ. The role of toll-like receptor (TLR) 2 and TLR4 in the host defense against disseminated candidiasis. J Infect Dis 2002; 185:14839. First citation in article

    11.  Forsyth CB, Mathews HL. Lymphocyte adhesion to Candida albicans. Infect Immun 2002; 70:51727. First citation in article

    12.  Brown GD, Herre J, Williams DL, Willment JA, Marshall AS, Gordon S. Dectin-1 mediates the biological effects of -glucans. J Exp Med 2003; 197:111924. First citation in article

    13.  Deva R, Shankaranarayanan P, Ciccoli R, Nigam S. Candida albicans induces selectively transcriptional activation of cyclooxygenase-2 in HeLa cells: pivotal roles of Toll-like receptors, p38 mitogen-activated protein kinase, and NF-B. J Immunol 2003; 171:304755. First citation in article

    14.  Jouault T, Bernigaud A, Lepage G, Trinel PA, Poulain D. The Candida albicans phospholipomannan induces in vitro production of tumour necrosis factor-alpha from human and murine macrophages. Immunology 1994; 83:26873. First citation in article

    15.  Suzuki T, Tsuzuki A, Ohno N, Ohshima Y, Yadomae T. Enhancement of IL-8 production from human monocytic and granulocytic cell lines, THP-1 and HL-60, stimulated with Malassezia furfur. FEMS Immunol Med Microbiol 2000; 28:15762. First citation in article

    16.  Marr KA, Koudadoust M, Black M, Balajee SA. Early events in macrophage killing of Aspergillus fumigatus conidia: new flow cytometric viability assay. Clin Diagn Lab Immunol 2001; 8:12407. First citation in article

    17.  Puig-Kroger A, Serrano-Gomez D, Caparros E, et al. Regulated expression of the pathogen receptor dendritic cell-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin in THP-1 human leukemic cells, monocytes, and macrophages. J Biol Chem 2004; 279:256808. First citation in article

    18.  Charrad RS, Gadhoum Z, Qi J, et al. Effects of anti-CD44 monoclonal antibodies on differentiation and apoptosis of human myeloid leukemia cell lines. Blood 2002; 99:2909. First citation in article

    19.  Rogers PD, Thornton J, Barker KS, et al. Pneumolysin-dependent and -independent gene expression identified by cDNA microarray analysis of THP-1 human mononuclear cells stimulated by Streptococcus pneumoniae. Infect Immun 2003; 71:208794. First citation in article

    20.  Cousins RJ, Blanchard RK, Popp MP, et al. A global view of the selectivity of zinc deprivation and excess on genes expressed in human THP-1 mononuclear cells. Proc Natl Acad Sci USA 2003; 100:69527. First citation in article

    21.  Affymetrix. Affymetrix genechip operating software user's guide. Available at: http://www.affymetrix.com/. Accessed 22 March 2005. First citation in article

    22.  Winer J, Jung CK, Shackel I, Williams PM. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem 1999; 270:419. First citation in article

    23.  Romagnoli G, Nisini R, Chiani P, et al. The interaction of human dendritic cells with yeast and germ-tube forms of Candida albicans leads to efficient fungal processing, dendritic cell maturation, and acquisition of a Th1 response-promoting function. J Leukoc Biol 2004; 75:11726. First citation in article

    24.  Torosantucci A, Chiani P, Cassone A. Differential chemokine response of human monocytes to yeast and hyphal forms of Candida albicans and its relation to the -1,6 glucan of the fungal cell wall. J Leukoc Biol 2000; 68:92332. First citation in article

    25.  Torosantucci A, Romagnoli G, Chiani P, et al. Candida albicans yeast and germ tube forms interfere differently with human monocyte differentiation into dendritic cells: a novel dimorphism-dependent mechanism to escape the host's immune response. Infect Immun 2004; 72:83343. First citation in article

    26.  Netea MG, Stuyt RJ, Kim SH, Van der Meer JW, Kullberg BJ, Dinarello CA. The role of endogenous interleukin (IL)18, IL-12, IL-1, and tumor necrosis factor in the production of interferon- induced by Candida albicans in human whole-blood cultures. J Infect Dis 2002; 185:96370. First citation in article

    27.  Mullick A, Elias M, Harakidas P, et al. Gene expression in HL60 granulocytoids and human polymorphonuclear leukocytes exposed to Candida albicans. Infect Immun 2004; 72:41429. First citation in article

    28.  Nawa T, Nawa MT, Adachi MT, et al. Expression of transcriptional repressor ATF3/LRF1 in human atherosclerosis: colocalization and possible involvement in cell death of vascular endothelial cells. Atherosclerosis 2002; 161:28191. First citation in article

    29.  Hesser BA, Liang XH, Camenisch G, et al. Down syndrome critical region protein1 (DSCR1), a novel VEGF target gene that regulates expression of inflammatory markers on activated endothelial cells. Blood 2004; 104:14958. First citation in article

    30.  Fong CW, Zhang Y, Neo SY, Lin SC. Specific induction of RGS16 (regulator of G-protein signalling 16) mRNA by protein kinase C in CEM leukaemia cells is mediated via tumour necrosis factor  in a calcium-sensitive manner. Biochem J 2000; 352:74753. First citation in article

    31.  Chen B, Shi Y, Smith JD, Choi D, Geiger JD, Mule JJ. The role of tumor necrosis factor  in modulating the quantity of peripheral blood-derived, cytokine-driven human dendritic cells and its role in enhancing the quality of dendritic cell function in presenting soluble antigens to CD4+ T cells in vitro. Blood 1998; 91:465261. First citation in article

    32.  Lechmann M, Krooshoop DJ, Dudziak D, et al. The extracellular domain of CD83 inhibits dendritic cell-mediated T cell stimulation and binds to a ligand on dendritic cells. J Exp Med 2001; 194:181321. First citation in article

    33.  Wisniewski HG, Hua JC, Poppers DM, Naime D, Vilcek J, Cronstein BN. TNF/IL-1-inducible protein TSG-6 potentiates plasmin inhibition by inter--inhibitor and exerts a strong anti-inflammatory effect in vivo. J Immunol 1996; 156:160915. First citation in article

    34.  Traynor TR, Kuziel WA, Toews GB, Huffnagle GB. CCR2 expression determines T1 versus T2 polarization during pulmonary Cryptococcus neoformans infection. J Immunol 2000; 164:20217. First citation in article

    35.  Ritter U, Meissner A, Ott J, Korner H. Analysis of the maturation process of dendritic cells deficient for TNF and lymphotoxin-alpha reveals an essential role for TNF. J Leukoc Biol 2003; 74:21622. First citation in article

    36.  Gauss KA, Bunger PL, Larson TC, Young CJ, Nelson-Overton LK, Siemsen DW, Quinn MT. Identification of a novel tumor necrosis factor -responsive region in the NCF2 promoter. J Leukoc Biol 2005; 77:26778. First citation in article

    37.  King AG, Johanson K, Frey CL, et al. Identification of unique truncated KC/GRO chemokines with potent hematopoietic and anti-infective activities. J Immunol 2000; 164:377482. First citation in article

    38.  Means TK, Hayashi F, Smith KD, Aderem A, Luster AD. The Toll-like receptor 5 stimulus bacterial flagellin induces maturation and chemokine production in human dendritic cells. J Immunol 2003; 170:516575. First citation in article

    39.  Filler SG, Pfunder AS, Spellberg BJ, Spellberg JP, Edwards JE Jr. Candida albicans stimulates cytokine production and leukocyte adhesion molecule expression by endothelial cells. Infect Immun 1996; 64:260917. First citation in article

    40.  Moratz C, Hayman JR, Gu H, Kehrl JH. Abnormal B-cell responses to chemokines, disturbed plasma cell localization, and distorted immune tissue architecture in Rgs1-/- mice. Mol Cell Biol 2004; 24:576775. First citation in article

    41.  Shi GX, Harrison K, Han SB, Moratz C, Kehrl JH. Toll-like receptor signaling alters the expression of regulator of G protein signaling proteins in dendritic cells: implications for G protein-coupled receptor signaling. J Immunol 2004; 172:517584. First citation in article

    42.  Mittelstadt PR, Ashwell JD. Cyclosporin A-sensitive transcription factor Egr-3 regulates Fas ligand expression. Mol Cell Biol 1998; 18:374451. First citation in article

    43.  Droin NM, Pinkoski MJ, Dejardin E, Green DR. Egr family members regulate nonlymphoid expression of Fas ligand, TRAIL, and tumor necrosis factor during immune responses. Mol Cell Biol 2003; 23:763847. First citation in article

    44.  Fernandez Pujol B, Lucibello FC, Zuzarte M, Lutjens P, Muller R, Havemann K. Dendritic cells derived from peripheral monocytes express endothelial markers and in the presence of angiogenic growth factors differentiate into endothelial-like cells. Eur J Cell Biol 2001; 80:99110. First citation in article

    45.  Li AL, Li HY, Jin BF, et al. A novel eIF5A complex functions as a regulator of p53 and p53-dependent apoptosis. J Biol Chem 2004; 279:492518. First citation in article

日期:2007年5月15日 - 来自[2005年第191卷第17期]栏目

Immune sensing of Candida albicans requires cooperative recognition of mannans and glucans by lectin and Toll-like receptors

1Department of Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

2Nijmegen University Center for Infectious Diseases, Nijmegen, The Netherlands.

3School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom.

4Department of Tumor Immunology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

5Department of Surgery, East Tennessee State University, Johnson City, Tennessee, USA.

    Abstract

The fungal pathogen Candida albicans has a multilayered cell wall composed of an outer layer of proteins glycosylated with N- or O-linked mannosyl residues and an inner skeletal layer of ?-glucans and chitin. We demonstrate that cytokine production by human mononuclear cells or murine macrophages was markedly reduced when stimulated by C. albicans mutants defective in mannosylation. Recognition of mannosyl residues was mediated by mannose receptor binding to N-linked mannosyl residues and by TLR4 binding to O-linked mannosyl residues. Residual cytokine production was mediated by recognition of ?-glucan by the dectin-1/TLR2 receptor complex. C. albicans mutants with a cell wall defective in mannosyl residues were less virulent in experimental disseminated candidiasis and elicited reduced cytokine production in vivo. We concluded that recognition of C. albicans by monocytes/macrophages is mediated by 3 recognition systems of differing importance, each of which senses specific layers of the C. albicans cell wall.

    Introduction

Invasive Candida albicans infections are a serious clinical threat in patients who are immunosuppressed or undergo major surgical procedures. Mortality associated with disseminated candidiasis can be as high as 30–40%, despite the availability of new antifungal drugs (1, 2). Host defense against systemic candidiasis relies mainly on the ingestion and elimination of C. albicans by cells of the innate immune system, especially neutrophils, monocytes, and macrophages (3-6). After activation of these leukocyte populations by C. albicans, release of proinflammatory cytokines such as TNF-, IL-1?, IL-6, and IFN- is the first step in the activation of anticandidal innate immune responses. Proinflammatory cytokines activate neutrophils and macrophages to phagocytose the fungus and to release toxic oxygen and nitrogen radicals, thus eliminating the invading pathogen (7, 8). The crucial role of proinflammatory cytokines for the host defense against C. albicans has been demonstrated by the increased susceptibility to candidiasis of knockout mice lacking these cytokines (9, 10). In contrast, antiinflammatory cytokines such as IL-4 and IL-10 have immunosuppressive effects (11). It is currently believed that the balance between pro- and antiinflammatory cytokine production is decisive in determining whether the host defense system is overwhelmed or able to eliminate the fungal pathogens (12).

Stimulation of proinflammatory cytokine production and the activation of innate immunity depend on accurate recognition of an invading pathogen. The basic strategy for recognizing pathogens by the cells of the innate immunity system consists of nonclonal recognition of conserved structures of microorganisms, called pathogen-associated molecular patterns (PAMPs), which are not present in mammalian cells. Several classes of pattern-recognition receptors (PRRs) recognize the various PAMPs, of which TLRs and C-type lectin receptors are probably the most important (13). We and others have recently identified TLR2 and TLR4 as important PRRs for C. albicans (14-17). The mannose receptor (MR) has also been implicated in the stimulation of cytokine production by C. albicans (18), and we have recently demonstrated that the C-type lectin DC-specific ICAM3-grabbing nonintegrin (DC-SIGN) mediates the uptake of C. albicans by dendritic cells (19).

The outer layer of the cell wall of C. albicans is enriched with mannoproteins, which represent 30–40% of the cell wall dry weight (20). The inner layer is composed of chitin and ?1,3- and ?1,6-glucan. Earlier studies have suggested that mannosylated proteins of C. albicans induce cytokine production (21), and C. albicans mannan reportedly interacts with TLR4 (22). It has been suggested that phospholipomannan of C. albicans is recognized by TLR2 and induces proinflammatory cytokine production (23), whereas ?-glucans were recognized by a complex of TLR2 and dectin-1 (24, 25). However, the relative contribution of these PAMPs and their respective receptors to cytokine stimulation by C. albicans is not known.

Despite the progress in understanding the interaction of some of the fungal PAMPs with leukocyte receptors, there is no integrated view of the mechanisms by which the immune system recognizes C. albicans. We hypothesized that recognition of C. albicans is a multiple-level process involving specific receptor systems that recognize each layer of the fungal cell wall. The skeleton of the C. albicans cell wall is mainly formed by chitin and ?1,3- and ?1,6-glucans, whereas the outer cell wall is enriched with proteins that are modified with both long-chain and highly branched N-linked mannosyl residues as well as short linear chains of O-linked mannosyl residues. Using a range of isogenic glycosylation mutants (26-29), ligand-specific blocking experiments, and PAMP receptor knockout mice, we show that recognition of C. albicans by monocytes/macrophages is a complex process involving multiple recognition systems and that these receptor systems recognize sequentially the various layers of the outer portion of the fungal cell wall.

    Results

Cell wall mutants of C. albicans.

We have taken a genetic approach to investigate how immune cells recognize a fungal pathogen. We have used well-defined stable isogenic mutants of C. albicans depleted in specific cell wall components to investigate cytokine responses of monocytes. The och1 mutant was defective in outer, branched N-linked glycans, and transmission electron microscopy (TEM) revealed a thicker cell wall lacking a fibrillar mannoprotein layer (ref. 29 and compare Figure 1B with Figure 1A). The mnt1 mnt2 mutant lacks the 4 terminal O-linked 1,2-mannosyl residues, but had normal N-mannan (24) and a fibrillar outer wall surface (Figure 1C). The pmr1 mutant has defects in both N- and O-linked mannosylation, and had a thinner and less fibrillar cell wall than the control strain (Figure 1D). The mnn4 mutant lacks phosphomannan (25) and was indistinguishable by TEM from that of wild-type cells (Figure 1E). Therefore this set of mutants provided a unique molecular biological tool to dissect the immune responses of monocytes.

   Figure 1

Cell wall morphology in the C. albicans strains used in this study. (A–E) TEM micrographs. (A) Wild-type strain NGY152 . (B) och1 null (strain NGY357; ref. 26) or doxycycline-regulated conditional (strain NGY361; ref. 29) mutants, which are defective in the branched outer N-linked mannosyl chains. (C) mnt1 mnt2 mutant (strain NGY337; ref. 27), which lacks 4 terminal O-linked 1,2-mannosyl residues. (D) pmr1 mutant (strain NGY355; ref. 26), which has gross defects in mannosylation, characterized by absence of phosphomannan and reduced O-linked and N-linked glycans. (E) mnn4 mutant (strain CDH15; ref. 28), which lacks phosphomannan. Scale bar: 100 nm. (F and G) Structure of the N- (F) and O-linked (G) glycans and the site of action of deleted gene products. Man, mannosyl; ?-GlcNAc, ? N-acetylglucosamine.

Mannan stimulates cytokines in a TLR4- and MyD88-dependent manner.

Purified mannan (a mixture of both N- and O-linked oligosaccharides) from C. albicans stimulated production of TNF and IL-6 in both human mononuclear cells (MNCs) and murine peritoneal macrophages (Figure 2A). The induction of TNF in murine peritoneal macrophages was significantly decreased in mice deficient in MyD88, demonstrating that a TLR-dependent mechanism was involved in this stimulation (Figure 2B). Indeed, the diminished stimulation of macrophages harvested from TLR4-defective ScCr mice demonstrated a TLR4-dependent stimulation of TNF by C. albicans mannan, whereas TLR2–/– mice produced normal amounts of cytokines (Figure 2B). Because purified mannan is a relatively weak cytokine stimulus compared with C. albicans, we investigated whether purified mannan would inhibit C. albicans–induced cytokine production by occupying the mannan-binding sites on leukocytes. The presence of purified mannan significantly inhibited the production of TNF and IL-6 stimulated by C. albicans, demonstrating that mannan-binding sites are important for the induction of cytokines by the yeast (Figure 2C). In control experiments, mannan had no effect on LPS-induced cytokine production (data not shown). Therefore, fungal mannan stimulated cytokine production, and pretreatment with purified mannan blocked the cytokine response induced by C. albicans yeast cells, suggesting an important role of mannan recognition receptors for the stimulation of cytokines by the fungus.

   Figure 2

The role of mannan for the cytokine stimulation by C. albicans. (A) Human MNCs or peritoneal macrophages from C57BL/6J mice were stimulated for 24 hours at 37°C with 50 μg/ml purified mannan from C. albicans. (B) Peritoneal macrophages from wild-type mice and mice deficient in MyD88, TLR2, or TLR4 were stimulated with mannan. TNF and IL-6 concentrations were measured in the supernatants by specific RIA and ELISA, respectively. Results are pooled data from 2 separate experiments with a total of 10 mice per group. (C) Purified mannan was preincubated for 1 hour at 37°C with human MNCs before stimulation with 1 x 106 yeast cells/ml. Supernatants were collected after additional incubation for 24 hours, and TNF and IL-6 were measured. (D) Human MNCs were stimulated for 24 hours at 37°C with whole cells of either wild-type C. albicans (strain NGY152; ref. 26), a mutant defective in the Golgi transporter pmr1 (strain NGY355; ref. 26), or a control strain in which a wild-type copy of PMR1 was introduced into the pmr1 mutant (strain NGY356; ref. 26). TNF and IL-6 concentrations were assessed by RIA and ELISA, respectively. Results (mean ± SD) are pooled triplicate data from 2 separate experiments with a total of 8 volunteers per group. *P < 0.05 versus wild-type.

The role of mannosyl residues for cytokine stimulation.

The role of global mannosylation of mannoproteins for the stimulation of cytokines by human MNCs was investigated using a pmr1 C. albicans mutant (26). The pmr1 mutant induced significantly less TNF and IL-6 than did the control strain in which PMR1 was reintroduced (Figure 2D). Therefore, normal cytokine stimulation is dependent on mannosylation of the cell wall.

To investigate the specific roles of N- and O-linked mannosyl residues for stimulation of cytokines by C. albicans, we compared the stimulation of cytokines by C. albicans strains defective in N-linked mannosyl residues (och1), O-linked mannosyl residues (mnt1 mnt2), and mannosylphosphate (mnn4) in human MNCs. The absence of O-linked mannosyl residues diminished cytokine release by 30% in the case of IFN-, whereas the production of TNF was only 15–20% lower (Figure 3, A and C). In contrast, the absence of N-linked mannosyl residues reduced cytokine release by 70% (Figure 3, A and C). No role of phosphomannan was observed for the induction of cytokine release (Figure 3). Phosphomannan is synthesized preferentially in stationary phase cells; however, no differences were found in cytokine induction by exponential or stationary phase yeast cells (data not shown). An mnn4 mutant in a serotype B background (30) was also unaffected in its cytokine-inducing properties (data not shown). Similar conclusions were drawn when a time course of the stimulation was performed (Figure 3, C and D). IL-6 release by the various mannosylation mutants displayed a production pattern identical to that of TNF (data not shown).

   Figure 3

The role of N- and O-linked mannosyl residues for cytokine stimulation by C. albicans. MNCs were stimulated for various time points with the various C. albicans strains: the wild-type parent NGY152 strain; the och1 mutant (strain NGY357; ref. 29), defective in N-linked mannan; the mnt1 mnt2 mutant (strain NGY337; ref. 27), defective in O-linked mannan; and the mnn4 mutant (strain CDH15; ref. 28), defective in phosphomannan. (A and C) C. albicans concentration-dependent stimulation curves for TNF (A) and IFN- (C) after stimulation for 24 hours. (B and D) Time-dependent stimulation curves for the 2 cytokines when MNCs were stimulated with the various C. albicans strains. Results (mean ± SD) are pooled triplicate data from 2 separate experiments with a total of 8 volunteers per group. *P < 0.05; **P < 0.01 versus wild-type.

Normal cytokine release was recovered in strains in which the deleted genes were reintegrated into their respective mutants (Figure 4, A and C). In strain TET-OCH1, the expression of OCH1 was regulated by placing the OCH1 open reading frame under the control of a promoter that was regulatable by tetracyclines. Growth in 20 μg/ml doxycycline switched off expression of OCH1 and resulted in a reduction of cytokine production to a similar level as the och1 mutant. Growth of TET-OCH1 in the absence of doxycycline resulted in expression of OCH1 and induced cytokine production similar to that of the wild-type strain CAI-4 (Figure 4B).

   Figure 4

Reintegration of the defective genes restores cytokine production. (A) MNCs were stimulated with the parent NGY152 strain, the N-linked mannosyl-defective C. albicans strain (och1; strain NGY357; ref. 29), and the complemented reintegrant och1/och1/OCH1 strain (strain NGY358). (B) Stimulation was also performed with the conditional doxycycline-dependent mutant (pTET/och1; strain NGY361; ref. 29) in both the absence and the presence of doxycycline (doxy). (C) Comparison of the mnt1 mnt2 mutant (strain NGY337; ref. 27), defective in O-linked mannosyl residues, with the mnt1 mnt2 + MNT1 reintegrant strain (strain NGY335; ref. 27). After 24 hours’ stimulation at 37°C, supernatants were collected, and cytokines were determined by RIA or ELISA. Results (mean ± SD) are pooled triplicate data from 2 separate experiments with a total of 8 volunteers per group. *P < 0.05; **P < 0.01 versus wild-type.

Similar results were obtained in experiments with live C. albicans cells. The live och1 strain induced only 29% of the TNF production induced by the control strain (0.36 ± 0.11 ng/ml versus 1.21 ± 0.23 ng/ml; P < 0.05), while the live mnt1 mnt2 strain stimulated 78% of the control TNF production (0.94 ± 0.21 ng/ml; P < 0.05). In contrast, PBMC stimulated with the mnn4 strain released normal amounts of TNF (1.79 ± 0.64 pg/ml; P = NS). A similar stimulation pattern was found for other cytokines (data not shown). Using live fungal cells, we assessed the morphology of the fungal elements during the assay (37°C incubation in RPMI medium in the absence of plasma or serum). Whereas formation of pseudohyphae was common, no fully developed hyphae were formed. Importantly, there were no differences in hyphal formation among the various mutant strains and the control strain during the incubation.

Recognition of O-linked and N-linked mannosyl residues by TLR4 and MR.

Specific anti-TLR4 and anti-MR blocking antibodies were used to investigate which PRR was involved in the recognition of mannosyl residues of the C. albicans cell wall. Blockade of either TLR4 or MR of human MNCs inhibited TNF production stimulated by the C. albicans wild-type strain NGY152. In contrast, differential blockade of cytokine release was observed in the case of the och1 and mnt1 mnt2 mutant strains. The TNF released by the PBMCs stimulated with the N-linked mannosylation-defective och1 mutant was inhibited by anti-TLR4, but not by the anti-MR antibody (Figure 5A), demonstrating that the MR recognizes N-linked mannosyl residues. The opposite was true for TNF stimulation by the O-linked mannosylation defective mnt1 mnt2 mutant, which was inhibited by anti-MR antibodies but not by anti-TLR4 antibodies, suggesting that TLR4 recognizes O-linked mannosyl residues (Figure 5A). This conclusion was confirmed in TLR4-deficient mice, which displayed lower TNF production after stimulation with CAI-4 and och1 strains but not after stimulation with the mnt1 mnt2 mutant (Figure 5B).

   Figure 5

Differential recognition of O- and N-linked mannosyl residues by TLR4 and MR. (A) Human MNCs were stimulated with the various C. albicans strains — the parent NGY152 strain; the och1 mutant (strain NGY357; ref. 29), defective in N-linked mannosylation; and the mnt1 mnt2 mutant (strain NGY337; ref. 27), defective in O-linked mannosylation — in the presence of monoclonal antibodies against TLR4 or MR or a isotype-matched control antibody. After 24 hours’ stimulation at 37°C, supernatants were collected, and TNF concentration was measured by RIA. Results are pooled triplicate data from 2 separate experiments with a total of 8 volunteers per group. (B) Murine peritoneal macrophages from TLR4+/+ C57BL/10J and TLR4–/– ScCr mice were stimulated with the various C. albicans strains: NGY152, the och1 mutant (NGY357; ref. 29), and the mnt1 mnt2 mutant (NGY337; ref. 27). After 24 hours’ stimulation at 37°C, supernatants were collected, and TNF levels were determined by RIA. Results (mean ± SD) are pooled data from 2 separate experiments with a total of 10 mice per group. *P < 0.05 versus stimulation in the presence of control antibodies (A) or versus TLR4+/+ mice (B).

The role of ?-glucan–dectin-1 interaction for cytokine stimulation.

Interaction of ?-glucans with dectin-1/TLR2 complexes has been shown to induce cytokine production, and DC-SIGN is an additional C-type lectin receptor that is able to recognize C. albicans. We hypothesized that interaction of C. albicans with either of these receptors may account for the residual cytokine production induced in the absence of the mannosyl residues. Accordingly, we used combinations of C. albicans mutant strains and receptor blockade and assessed TNF release by the och1 mutant in TLR4–/– mice and by the mnt1 mnt2 strain in the presence of anti-MR antibodies. In these 2 situations, the signals induced by both N-linked mannosyl/MR and O-linked mannosyl/TLR4 complexes were absent. The residual cytokine production stimulated by C. albicans in these 2 experimental conditions was completely blocked by laminarin, a ligand of dectin-1 that is rich in ?1,3-glucan (Figure 6). In contrast, no role for DC-SIGN could be demonstrated using specific blocking antibodies (data not shown). Interestingly, when dectin-1–blocking experiments were performed with either heat-killed or live C. albicans microorganisms, laminarin had a much stronger inhibitory effect on cytokines induced by heat-killed C. albicans yeasts compared with live fungi (data not shown). This finding strongly sustains the recent findings of Gantner et al., who reported that the ?-glucans of the cell wall in live cells are shielded from recognition by the mannan layers but become exposed in heat-killed C. albicans (31).

   Figure 6

The role of ?-glucan/dectin-1 interaction for cytokine stimulation by C. albicans. The role of ?-glucan/dectin-1 interaction for C. albicans–induced TNF was investigated using 2 approaches with combinations of mutant C. albicans strains and receptor blockade: (A) stimulation with the och1 NGY357 strain (29) in TLR4–/– mice and (B) stimulation with mnt1 mnt2 NGY337 strain (27) in the presence of anti-MR antibodies in human MNCs. In both situations, the signals induced by N-linked mannosyl/MR and O-linked mannosyl/TLR4 complexes were deficient. The residual cytokine production stimulated by C. albicans in these 2 experimental conditions was completely blocked by laminarin, a ligand of dectin-1. Results (mean ± SD) are pooled data from 2 separate experiments with a total of 10 mice per group (A) or 8 human volunteers (B). *P < 0.05; **P < 0.01; ***P < 0.001 versus wild-type.

The role of mannosyl residues in the virulence of C. albicans.

Published experiments from our groups have established the role of mannosyl residues in the virulence of C. albicans. We have shown that the och1, pmr1, and mnt1 mnt2 strains are attenuated in virulence in a mouse model (26, 27), but that the mnn4 mutant is unaffected in virulence (28). We extended this in the present study by characterizing the course of disseminated candidiasis and cytokine production in mice infected with the och1 C. albicans strain. Compared with the wild-type strain, the och1 strain induced significantly less mortality in a model of disseminated candidiasis in mice (Figure 7A) and had a reduced fungal load in the organs of the mice (Figure 7B). However, viable cells were still present in kidneys with little sign of disease. Virulence was fully recovered in the control strain that contained a reintegrated copy of OCH1 (Figure 7). These virulence parameters were paralleled by significantly decreased cytokine levels in the kidneys of the mice infected with the och1 C. albicans mutant (Figure 7C).

   Figure 7

The role of N-linked mannosyl residues in virulence and in vivo cytokine production of C. albicans. (A) Survival of mice infected intravenously with 5 x 106 CFU of wild-type C. albicans, the och1 strain, or the reintegrant control strain. (B) Fungal burden of C. albicans in the kidneys of mice infected intravenously with 1 x 105 CFU of the strains described in A. (C) Cytokine levels on day 3 in the kidneys of mice infected with the strains described in A. Data are presented as means ± SD. Results are pooled data from 2 separate experiments with a total of 10 mice per group. *P < 0.05 versus wild-type.

    Discussion

In the present study, we have shown that 3 components of the cell wall of a pathogenic fungus, N-linked mannans, O-linked mannans, and ?-glucans, are involved in the recognition by monocytes/macrophages and for the subsequent induction of pro- and antiinflammatory cytokine release. To investigate the role of cell wall mannosyl groups for the recognition of C. albicans, we used isogenic mutant strains of C. albicans with specific defects in the mannosylation of cell wall proteins. A gross defect in protein mannosylation was investigated using C. albicans Pmr1p, which encodes a Mn2+/Ca2+ transporter necessary for the activity of several Golgi-bound mannosyl transferases and shows defects in both N-linked and O-linked mannosylation (29). The role of phosphomannan was investigated in the mnn4 mutant (28), while the role of O-linked mannosylation was assessed using a mnt1 mnt2 mutant strain lacking 2 partially redundant 1,2-mannosyl transferases that are required to add the second and third mannose residues to a linear oligomannoside (27). Finally, the effects of N-linked mannosylation were investigated in the och1 C. albicans strain, which lacks an 1,6-mannosyl transferase that is required for initiation of the synthesis of the branched outer mannan chains (29).

We demonstrated that N-linked and O-linked mannosyl groups of glycoproteins of the outer surface of the cell wall were responsible for most of the cytokine-stimulating activity by the yeast cell. This was achieved through the specific interaction of the N-linked mannosyl residues with MR and of the O-linked mannosyl residues with TLR4. However, the mannosylphosphate fraction of the N-linked glycan played practically no role in this process. The residual cytokine production stimulated by C. albicans strains lacking mannosyl residues was mediated by the interaction of ?-glucan with dectin-1, probably in cooperation with TLR2, as discussed below. Thus 3 C. albicans PAMPs cooperate in the activation of the innate immune response, and recognition is a multilevel process involving cooperation between TLRs (TLR4, TLR2) and lectin receptors (MR, dectin-1).

Mannoproteins, ?1,2-oligomannosides, phospholipomannan, and ?-glucans have all been implicated individually in cytokine stimulation induced by C. albicans (21, 32-34). The ?1,3-glucans are mainly situated in the interior layer of the cell wall of C. albicans, and living yeast cells do not display large amounts of glucans at their surface. ?-glucans become accessible only when the surface mannan coat is removed or damaged by killing, as is the case in zymosan particles that are chemically denuded of mannan (35), or at the site of budding scars in C. albicans yeasts, which are absent from hyphal cells (31). In contrast, - and ?-linked mannosyl residues are abundant at the surface of the fungus. We found that -linked mannan is critically involved in cytokine stimulation. The ?-linked mannosyl residues are present in the acid-labile and acid-stable fractions of N-linked mannans in serotype A strains, in the acid-labile fraction of serotype B strains, and in the structure of phospholipomannan (36). However, cytokine induction was not affected in assays with mnn4 mutants in either serotype A or serotype B backgrounds (29). The cell wall is therefore a multilayered structure, and it is the external, heavily -linked, mannosylated portion of the cell wall that is most strongly recognized by monocytes.

Mannoproteins have been previously implicated as important cytokine stimuli (21) — our study provides a refined molecular description of this process. The results of our 3 independent approaches support this conclusion. First, we showed that purified mannan isolated from C. albicans stimulated cytokine production in a manner dependent partly on MyD88, an intracellular TLR adaptor molecule (37), and on TLR4. This extends previous studies that also suggested TLR4-dependent cytokine production by mannan (22). Second, we demonstrated that mannan-binding sites on the surface of leukocytes were important for recognition of the yeast cell surface (5). Third, mutants lacking N- or O-linked mannosyl residues were markedly affected in their ability to induce cytokines in monocytes.

We demonstrated that a global defect in the mannosylation of cell wall mannoproteins due to in the absence of the pmr1 Golgi transporter resulted in strongly reduced cytokine induction. Perhaps surprisingly, considering the reported immunostimulatory effects attributed to mannosylphosphate (38), a mannosylphosphate-deficient mnn4 mutant induced normal cytokine production. This was in agreement with the previous observation that this mutant is recognized normally by macrophages (28). In contrast, approximately 70% less cytokine production was stimulated by och1 mutants lacking branched N-linked mannan, and 30% less cytokine production was seen using a C. albicans mutant with truncated O-linked mannan. A similar difference in cytokine production as found between heat-killed C. albicans strains was seen with the live microorganisms. This is an important observation, because heat-killed C. albicans was present exclusively as yeasts, whereas large numbers of pseudohyphae were present in the preparations using live C. albicans. Therefore, mannans are the most important structure for the induction of cytokines by both live and heat-killed C. albicans, in accord with the localization of these epitopes at the exterior face of the fungal cell wall and with the modifications of the superficial cell wall structure visible by electron microscopy (Figure 1).

In addition to demonstrating the role of protein mannosylation for C. albicans–induced cytokine production, we also identified the PRRs involved in the specific recognition of N-linked and O-linked mannosyl residues. Using a combination of mannosyl-defective C. albicans strains, anti-receptor blocking antibodies, and knockout mice, we demonstrated that MR recognized the highly branched N-linked mannosyl chains whereas TLR4 bound and recognized the linear O-linked mannosyl chains. The role of MR (39, 40) and TLR4 (14, 22) in the recognition of C. albicans has already been proposed, but to our knowledge the C. albicans PAMPs recognized by these receptors have not previously been identified. We have demonstrated previously that differential activation of PRRs such as TLR2 and TLR4 by C. albicans activates specific responses (14, 15), and we and others proposed that this was a universal mechanism of modulation of the innate host defense (13, 41). In the present study we showed that the innate immune system was able to recognize specific structures of the fungal cell wall, and we are currently investigating how this recognition is translated into differentiated immune responses.

Using combinations of mutant C. albicans strains and receptor blockade (testing the och1 strain in TLR4–/– mice and the mnt1 mnt2 strain in the presence of anti-MR antibodies), we also demonstrated that the residual cytokine production stimulated by C. albicans in the absence of signals mediated by mannosyl residues was mediated by the ?-glucan receptor dectin-1. Dectin-1 has been reported to be the major receptor for ?-glucan (42), and recent studies have demonstrated that it forms a receptor complex with TLR2, amplifying its effects (24, 25). Moreover, TLR2-independent activity of dectin-1 has also been recently reported (43). In addition, the recognition of yeasts, but not hyphae, by dectin-1 has been proposed to represent an escape mechanism of the fungus (31).

The differences in pattern recognition and cytokine production among different C. albicans strains were reflected by significant differences in the virulence in in vivo models of disseminated candidiasis. In earlier studies we have demonstrated reduced virulence of the pmr1, och1, and mnt1 mnt2 strains (26, 27). In the present study we confirmed a significant reduction in the virulence of the och1 strain and showed that this strain induced fewer cytokines in vivo (Figure 7). However, the experimental model of candidiasis is more complex than the in vitro experiments. Whereas stimulation of monocytes/macrophages in vitro specifically investigates the role of mannosyl residues for the recognition of C. albicans, the outcome of the experimental infection is influenced by multiple mechanisms, including pattern recognition and cytokine production as well as other factors such as adherence to host endothelial cells and growth rate of the various mutants. The och1 strain not only was defective in cytokine induction, as shown in the present study, but also displays hypersensitivity to agents that perturb the cell wall (29), which may be reflected in a lower resistance to candidacidal mechanisms. Thus a combination of these factors most likely contributes to the lower virulence of the och1 C. albicans mutant.

In conclusion, we show that C. albicans induced cytokine stimulation in mammalian MNCs via 3 pathways, each recognizing 1 of the multilayered structures of the fungal cell wall: N-linked mannosyl polymers are recognized by MR, O-linked chains by TLR4, and ?-glucans by dectin-1/TLR2. Phosphomannan is apparently not involved in pro- or antiinflammatory cytokine induction. The specific activity of the cytokine induction response to these cell wall components reflects their abundance and accessibility in the C. albicans yeast cell wall. This study is the first to our knowledge that describes in totality the recognition pathways of a fungal pathogen and can serve as model for future studies of the innate recognition of other microorganisms.

    Methods

Animals.

TLR4-deficient C57BL/ScCr mice were from a local colony at Radboud University Nijmegen, and control TLR4-competent C57BL/10J mice and C57BL/6J mice were obtained from The Jackson Laboratory. MyD88–/– mice and TLR2–/– mice on a C57BL/6 background were kindly provided by S. Akira (Tokyo University, Tokyo, Japan). All mice weighed 20–25 g and were 6–8 weeks old. The mice were fed sterilized laboratory chow (Hope Farms) and water ad libitum. The experiments were approved by the ethics committee on animal experiments of Radboud University Nijmegen.

C. albicans strains and growth conditions.

Homozygous null mutants in glycosylation genes were constructed in the C. albicans CAI-4 serotype A background by targeted gene disruption (44). Control strains were used in which the wild-type genes, under the control of their own promoters, and the URA3 selectable marker were reintroduced at the neutral RPS1 locus via the CIp10 integrative plasmid (45). Parental strain CAI-4 was also transformed with empty CIp10, generating strain NGY152, making all strains isogenic with regard to Ura status. In addition, a conditional och1 mutant was also used in which 1 remaining functional allele was regulated by the tetracycline/doxycycline–repressable TET promoter (strain NGY361; ref. 29). Mutants had defects in outer chain N-mannosylation (och1; strain NGY357; ref. 29), O-mannosylation (mnt1 mnt2; strain NGY337; ref. 27), and phosphomannan biosynthesis (mnn4; strain CDH15; ref. 28) or were downregulated in glycosylation due to low levels of Mn2+ in the Golgi (pmr1; strain NGY355; ref. 26). C. albicans was grown with continuous shaking at 200 rpm at 30°C in Sabouraud broth (1% mycological peptone/4% glucose) overnight, transferred to fresh medium, and incubated for 4 hours. All mutant strains showed similar morphology (yeasts and hyphae) to the control C. albicans strain, either when incubated in growth medium or together with PBMCs, as described previously in detail (26-29). The cells were harvested by centrifugation, and the pellets were washed twice in 20 ml sterile PBS and resuspended to a density of 1 x 108 cells/ml before heat-killing at 56°C for 1 hour. The TET-OCH1 strain was pregrown overnight in the presence of 20 μg/ml doxycycline. In separate experiments, live C. albicans yeast cells were washed and resuspended in RPMI 1640 at a concentration of 1 x 106 CFU/ml and used for the stimulation of cytokine production. The mnt1 mnt2, och1, and pmr1 mutant strains tended to aggregate more easily in culture medium, and we cannot completely exclude a certain degree of aggregation even after disrupting them by vigorous vortexing. This may have resulted in some underestimation of the yeast number in the suspension containing these mutant strains. In contrast, the mnn4 mutant did not aggregate.

Freeze substitution TEM.

Midexponential phase yeast cells were grown in yeast extract, peptone, and dextrose medium and harvested by centrifugation, and the pellets were resuspended in 1% agarose and transferred to flat specimen carriers. The samples were frozen in liquid nitrogen at high pressure using a Leica EM PACT high-pressure freezer (Leica Microsystems). Freeze substitution of the frozen cells was carried out in an automatic temperature-controlled freeze substitution system (AFS; Leica Microsystems) in dried acetone containing 1% OsO4 at –90°C for 48 hours. The samples were gradually warmed to –30°C and then processed in a Lynx tissue processor to finish in acetone/resin at a ratio of 1:2. The samples were transferred to a Lynx tissue processor to embed in TAAB812 epoxy resin (TAAB Laboratory Equipment Ltd.). Ultra-thin sections (60 nm) were cut with a Leica ultracut E, and the sections were stained with uranyl acetate and lead citrate. Samples were imaged in a Philips CM10 transmission microscope (FEI UK Ltd.), and the images were recorded with a Gatan Bioscan 792 (Gatan).

Fungal carbohydrate polymers.

The mannan from C. albicans was isolated as previously described by Kogan et al. (46). Briefly, 100 g wet wt of yeast biomass was suspended in 400 ml 2% (wt/vol) KOH and heated for 1 hour at 100°C. Insoluble residues were separated by centrifugation, and mannan was precipitated from supernatant with Fehling’s reagent. The sedimented mannan-copper complex was dissolved in a minimum volume of 3 M HCl and added dropwise to methanol/acetic acid at a ratio of 8:1 (vol/vol). The procedure of dissolution and precipitation was repeated twice. Finally, the sediment was separated, dissolved in distilled water, and dialysed for 24 hours. Mannan contained no nitrogen as determined by elementary analysis. Laminarin was purchased from Sigma-Aldrich. The mannan and laminarin were chemically characterized to confirm their molecular weight and chemical structure (47, 48) and were assayed to confirm the absence of endotoxin.

Antibodies.

The monoclonal anti-TLR4 HTA125 antibody was a kind gift of K. Miyake (Saga Medical School, Saga, Japan). The monoclonal mouse anti-human MR antibody and the isotype-matched IgG antibody used as a control in all experiments were purchased from Sigma-Aldrich.

Stimulation of cytokine production in human MNCs.

Isolation of MNCs was performed as described previously (49). Venous blood was collected from cubital veins of 8 healthy volunteers. All volunteers gave informed consent prior to participating in the study. Samples of 5 x 105 MNCs in a 100-μl volume were added to round-bottomed 96-well plates (Greiner Bio-One) and incubated for 24 hours with 100 μl of the various strains of live or heat-killed (30 minutes at 56°C) C. albicans at a concentration 1 x 106 yeast cells/ml unless otherwise indicated. In receptor-blocking studies, MNCs were preincubated for 1 hour at 37°C with the various monoclonal antibodies (anti-TLR4, anti-MR, or control IgG; 10 μg/ml) before stimulation with C. albicans. After 24 hours’ incubation at 37°C, the MNC/C. albicans cell suspensions were centrifuged, and the supernatants were collected and stored at –70°C until assayed. Human TNF- concentrations were determined by specific RIAs as described previously (50). IL-6, IL-10, and IFN- concentrations were measured by commercial ELISA kits (Sanquin).

Cytokine production by murine peritoneal macrophages.

Resident peritoneal macrophages from the various mouse strains were harvested by injecting 4 ml sterile PBS containing 0.38% sodium citrate (8). After centrifugation and washing, the cells were resuspended in RPMI 1640. Cells were cultured in 96-well plates and stimulated with 100 μl of the various C. albicans strains for 24 hours at 37°C (50). Murine IL-1, IL-1?, and TNF- were determined by specific RIAs (detection limit, 20 pg/ml) (51).

C. albicans infection model.

We used a C. albicans infection model as described previously (15). Briefly, 1 x 105 (for survival experiments) or 1 x 105 CFUs (for fungal burden) of C. albicans strains (wild-type, och1, or the reintegrant strain) were injected i.v. into mice on day 0. Survival was assessed daily. On days 3, 7, and 14, kidneys and livers of subgroups of mice were aseptically removed, weighed, and homogenized in sterile saline in a tissue grinder. The number of viable C. albicans cells was determined by plating serial dilutions on Sabouraud dextrose agar plates. The colonies were counted after 24 hours at 37°C, and results were expressed as CFU/g tissue. In addition, tissue homogenates were centrifuged, and cytokines were measured in the supernatant by ELISA (see above).

Statistics.

The human experiments were performed using triplicate samples in 2 experiments with a total of 8 volunteers. The mouse experiments were performed twice in 10 mice per group. The differences between groups were analyzed by Mann-Whitney U test. The level of significance between groups was set at P < 0.05. Data are given as means ± SD.

    Acknowledgments

We thank D. Singleton and K. Hazen for the mnn4 serotype B strain. This study was partly supported by a Vidi grant of the Netherlands Organization for Scientific Research to M.G. Netea. N.A.R. Gow, A.J.P. Brown, and F.C. Odds acknowledge financial support from the Wellcome Trust (grants 06324 and 72263). D.L. Williams was supported in part by Public Health Service grants GM53522 from the National Institute of General Medical Sciences and AI45829 from the National Institute of Allergy and Immunology.

References

Pfaller, M.A. et al. 1999. . International surveillance of blood stream infections due to Candida species in the European SENTRY Program: species distribution and antifungal susceptibility including the investigational triazole and echinocandin agents. Diagn. Microbiol. Infect. Dis. 35::19-25.

Edmond, M.B. et al. 1999. . Nosocomial bloodstream infections in United States hospitals: a three-year analysis. Clin. Infect. Dis. 29::239-244.

Van ‘t Wout, J.W., Linde, I., Leijh, P.C.J., and Van Furth, R. 1988. . Contribution of granulocytes and monocytes to resistance against experimental disseminated Candida albicans infections. Eur. J. Clin. Microbiol. Infect. Dis. 7::736-741.

Kullberg, B.J., Van ‘t Wout, J.W., and Van Furth, R. 1990. . Role of granulocytes in enhanced host resistance to Candida albicans induced by recombinant interleukin-1. Infect. Immun. 58::3319-3324.

Marodi, L., Korchak, H.M., and Johnston, R.B. Jr. 1991. . Mechanisms of host defense against Candida species. 1. Phagocytosis by monocytes and monocyte-derived macrophages. J. Immunol. 146::2783-2789.

Qian, Q., Jutila, M.A., Van Rooijen, N., and Cutler, J.E. 1994. . Elimination of mouse splenic macrophages correlates with increased susceptibility to experimental disseminated candidiasis. J. Immunol. 152::5000-5008.

Djeu, J.Y. 1990. . Role of tumor necrosis factor and colony-stimulating factors in phagocyte function against Candida albicans. Diagn. Microbiol. Infect. Dis. 13::383-386.

Kullberg, B.J., Van ‘t Wout, J.W., Hoogstraten, C., and Van Furth, R. 1993. . Recombinant interferon- enhances resistance to acute disseminated Candida albicans infection in mice. J. Infect. Dis. 168::436-443.

Netea, M.G. et al. 1999. . The increased susceptibility of TNFLT double knock-out mice to systemic candidiasis is due to defective recruitment and phagocytosis by neutrophils. J. Immunol. 163::1498-1505.

Kaposzta, R., Tree, P., Marodi, L., and Gordon, S. 1998. . Characteristics of invasive candidiasis in gamma interferon- and interleukin-4-deficient mice: role of macrophages in host defense against Candida albicans. Infect. Immun. 66::1708-1717.

Tonnetti, L. et al. 1995. . Interleukin-4 and -10 exacerbate candidiasis in mice. Eur. J. Immunol. 25::1559-1565.

Romani, L. 2004. . Immunity to fungal infections. Nat. Rev. Immunol. 4::1-13.

Netea, M.G., Van der Graaf, C., Van der Meer, J.W.M., and Kullberg, B.J. 2004. . Toll-like receptors and the host defense against microbial pathogens: bringing specificity to the innate-immune system. J. Leukoc. Biol. 75::749-755.

Netea, M.G. et al. 2002. . The role of toll-like receptor (TLR) 2 and TLR4 in the host defense against disseminated candidiasis. J. Infect. Dis. 185::1483-1489.

Netea, M.G. et al. 2004. . Toll-like receptor 2 inhibits cellular responses against Candida albicans through pathways mediated by IL-10 and CD4+CD25+ regulatory T cells. J. Immunol. 172::3712-3718.

Bellocchio, S. et al. 2004. . The contribution of Toll-like/IL-1 receptor superfamily to innate and adaptive immunity to fungal pathogens in vivo. J. Immunol. 172::3059-3069.

Villamon, E. et al. 2004. . Toll-like receptor-2 is essential in murine defenses against Candida albicans infections. Microbes Infect. 6::1-7.

Yamamoto, Y., Klein, T.W., and Friedman, H. 1997. . Involvement of mannose receptor in cytokine interleukin-1beta (IL-1beta), IL-6, and granulocyte-macrophage colony-stimulating factor responses, but not in chemokine macrophage inflammatory protein 1beta (MIP-1beta), MIP-2, and KC responses, caused by attachment of Candida albicans to macrophages. Infect. Immun. 65::1077-1082.

Cambi, A. et al. 2003. . The C-type lectin DC-SIGN (CD209) is an antigen-uptake receptor for Candida albicans on dendritic cells. Eur. J. Immunol. 33::532-538.

Klis, F.M., de Groot, P., and Hellingwerf, K. 2001. . Molecular organization of the cell wall of Candida albicans. Med. Mycol. 39(Suppl. 1)::1-8.

Vecchiarelli, A., Puliti, M., Torosantucci, A., Cassone, A., and Bistoni, F. 1991. . In vitro production of tumor necrosis factor by murine splenic macrophages stimulated with mannoprotein constituents of Candida albicans cell wall. Cell Immunol. 134::65-76.

Tada, H. et al. 2002. . Saccharomyces cerevisiae- and Candida albicans-derived mannan induced production of tumor necrosis factor alpha by human monocytes in a CD14- and Toll-like receptor 4-dependent manner. Microbiol. Immunol. 2002::503-512.

Jouault, T. et al. 2003. . Candida albicans phospholipomannan is sensed through toll-like receptors. J. Infect. Dis. 188::165-172.

Gantner, B.N., Simmons, R.M., Canavera, S.J., Akira, S., and Underhill, D.M. 2003. . Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor 2. J. Exp. Med. 197::1107-1117.

Brown, G.D. et al. 2003. . Dectin-1 mediates the biological effects of beta-glucans. J. Exp. Med. 197::1119-1124.

Bates, S. et al. 2005. . Candida albicans Pmr1p, a secretory pathway P-type Ca2+/Mn2+-ATPase, is required for glycosylation and virulence. J. Biol. Chem. 280::23408-23415.

Munro, C.A. et al. 2005. . Mnt1p and Mnt2p of Candida albicans are partially redundant alpha-1,2-mannosyltransferases that participate in O-linked mannosylation and are required for adhesion and virulence. J. Biol. Chem. 280::1051-1060.

Hobson, R.P. et al. 2004. . Loss of cell wall mannosylphosphate in Candida albicans does not influence macrophage recognition. J. Biol. Chem. 279::39628-39635.

Bates, S. et al. 2006. . Outer chain N-glycans are required for cell wall integrity and virulence of Candida albicans. J. Biol. Chem. 281::90-98.

Singleton, D.R., Masuoka, J., and Hazen, K.C. 2005. . Surface hydrophobicity changes of two Candida albicans serotype B mnn4 delta mutants. Eukaryotic Cell. 4::639-648.

Gantner, B.N., Simmons, R.M., and Underhill, D.M. 2005. . Dectin-1 mediates macrophage recognition of Candida albicans yeasts but not filaments. EMBO J. 24::1277-1286.

Jouault, T., Bernigaud, A., Lepage, G., Trinel, P.A., and Poulain, D. 1994. . The Candida albicans phospholipomannan induces in vitro production of tumour necrosis factor-alpha from human and murine macrophages. Immunology. 83::268-273.

Jouault, T. et al. 1995. . ?-1,2-linked oligomannosides from Candida albicans act as signals for tumor necrosis factor alpha production. Infect. Immun. 63::2378-2381.

Torosantucci, A., Chiani, P., and Cassone, A. 2000. . Differential chemokine response of human monocytes to yeast and fungal forms of Candida albicans and its relation to the beta-1,6 glucan of the fungal cell wall. J. Leukoc. Biol. 68::923-932.

Poulain, D., and Jouault, T. 2004. . Candida albicans cell wall glycans, host receptors and responses: elements for a decisive cross-talk. Curr. Opin. Microbiol. 7::342-349.

Gow, N.A., Brown, A.J., and Odds, F.C. 2002. . Fungal morphogenesis and host invasion. Curr. Opin. Microbiol. 5::366-371.

Serbina, V. et al. 2003. . Sequential MyD88-independent and -dependent activation of innate immune responses to intracellular bacterial infection. Immunity. 19::891-898.

Cutler, J.E. 2001. . N-glycosylation of yeast, with emphasis on Candida albicans. Med. Mycol. 39S::75-86.

Newman, S.L., and Holly, A. 2001. . Candida albicans is phagocytosed, killed, and processed for antigen presentation by human dendritic cells. Infect. Immun. 69::6813-6822.

Porcaro, I., Vidal, M., Jouvert, S., Stahl, P.D., and Giaimis, J. 2003. . Mannose receptor contribution to Candida albicans phagocytosis by murine E-clone J774 macrophages. J. Leukoc. Biol. 74::206-215.

Underhill, D.M., and Ozinsky, A. 2002. . Toll-like receptors: key mediators of microbe detection. Curr. Opin. Immunol. 14::103-110.

Brown, G.D. et al. 2002. . Dectin-1 is a major beta-glucan receptor on macrophages. J. Exp. Med. 196::407-412.

Rogers, N.C. et al. 2005. . Syk-dependent cytokine induction by Dectin-1 reveals a novel pattern recognition pathway for C type lectins. Immunity. 22::507-517.

Fonzi, W.A., and Irwin, M.Y. 1993. . Isogenic strain construction and gene mapping in Candida albicans. Genetics. 134::717-728.

Brand, A., MacCallum, D.M., Brown, A.P.J., Gow, N.A.R., and Odds, F.C. 2004. . Ectopic expression of URA3 can influence the virulence phenotypes and proteome of Candida albicans but can be overcome by targeted re-integration of URA3 at the RPS10 locus. Eukaryot. Cell. 3::900-909.

Kogan, G., Pavliak, V., and Masler, L. 1988. . Structural studies of mannans from the cell walls of the pathogenic yeasts Candida albicans serotypes A and B and Candida parapsilosis. Carbohydr. Res. 172::243-253.

Mueller, A. et al. 2000. . The influence of glucan polymer structure and solution conformation on binding to (1-->3)-beta-D-glucan receptors in a human monocyte-like cell line. Glycobiology. 10::339-346.

Lowman, D.W., Ferguson, D.A., and Williams, D.L. 2003. . Structural characterization of (1-->3)-beta-D-glucans isolated from blastospore and hyphal forms of Candida albicans. Carbohydr. Res. 338::1491-1496.

Endres, S., Ghorbani, R., Lonnemann, G., Van der Meer, J.W.M., and Dinarello, C.A. 1988. . Measurement of immunoreactive interleukin-1 beta from human mononuclear cells: optimization of recovery, intrasubject consistency, and comparison with interleukin-1 alpha and tumor necrosis factor. Clin. Immunol. Immunopathol. 49::424-438.

Drenth, J.P.H. et al. 1995. . Endurance run increases circulating IL-6 and IL-1ra but downregulates ex vivo TNF- and IL-1? production. J. Appl. Physiol. 79::1497-1503.

Netea, M.G. et al. 1996. . Low-density-lipoprotein receptor deficient mice are protected against lethal endotoxinemia and severe Gram-negative infections. J. Clin. Invest. 97::1366-1372.

 

日期:2007年5月11日 - 来自[2006年第116卷第6期]栏目
循环ads

PCR Protocol for Specific Identification of Candida nivariensis, a Recently Described Pathogenic Yeast

    Department of Microbiology, Hospital Universitario N. S. de Candelaria, Santa Cruz de Tenerife, Canary Islands, Spain
    Research Institute, Hospital Universitario N. S. de Candelaria, Santa Cruz de Tenerife, Canary Islands, Spain
    Department of Cellular Biology & Microbiology, University of La Laguna, Canary Islands, Spain
    Unitat de Microbiologia, Facultat de Medicina i Ciencias de la Salut, Universitat Rovira i Virgili, Reus, Tarragona, Spain
    Department of Public Health, School of Medicine, University of La Laguna, Tenerife, Spain

    ABSTRACT

    Candida nivariensis is a recently described pathogenic yeast closely related to Candida glabrata. We developed a specific set of oligonucleotide primers based on the internal transcribed spacer regions of the rRNA gene for the rapid identification of C. nivariensis. PCR with these primers amplified a 206-bp amplicon from C. nivariensis.

    TEXT

    Invasive fungal infections are a major medical problem, particularly among immunocompromised hosts (9). The management of invasive fungal infections has been hampered by the inability to diagnose the infection at an early stage of disease. However, diagnosis of these fungal infections remains difficult, since the only clinical sign of infection may be a prolonged fever that is refractory to antibacterial treatment. In recent years, efforts have been made to develop molecular biology-based methods for rapid diagnosis, which is crucial for the treatment and recovery of patients suffering from systemic candidiasis (8).

    Yeasts are usually identified through a combination of morphological features, ability to ferment selected sugars, and performance of assimilation reactions on a relatively large number of carbon and nitrogen compounds (6). Molecular studies have shown that it is not uncommon for different strains of a species to vary somewhat in their fermentation and assimilation profiles, which can lead to misidentifications (10, 4). Molecular approaches are more promising than phenotypic methods for the rapid detection and identification of pathogenic organisms (2, 3, 7, 11, 12). The recently described species Candida nivariensis differs somewhat from other known species in relation to physiological reactions (1).

    A total of 35 yeast isolates, including the three available isolates of C. nivariensis, other relevant pathogenic yeasts, and four reference strains, were included in this study. The three isolates of C. nivariensis were identified as was described previously (1).

    Extraction of nuclear DNA of the isolates was performed as previously described (5). Two oligonucleotides (NIV-F [AGCTCATCCTGGTTAGTTTCG] and NIV-R [CCCTCTTCGTTTGTGTTTGT]) were designed after comparison of different yeast rRNA sequences from the GenBank database. Nucleotide-nucleotide BLAST (blastn) comparisons showed that the only sequences that showed 100% identities with both primers were the internal transcribed spacer (ITS) sequence of one unidentified isolate deposited in the database (accession number AY787833.1) and the ITS sequences from the three isolates of C. nivariensis (1). The set was synthesized by Roche Diagnostics.

    PCRs were carried out in 50-μl reaction volumes containing about 0.05 ng of extracted DNA added to the PCR mixture consisting of 1x reaction buffer [16 mM (NH4)2SO4, 67 mM Tris-HCl (pH 8.8)], containing 0.2 mM of each of the deoxynucleoside triphosphates (Promega Corp., Madison, Wis.), 1.5 mM MgCl2, 10 pmol of each primer, and 1.25 U of Taq polymerase (Bioline). DNA amplification was performed in a GeneAmp PCR system 9700 thermocycler (PE Applied Biosystems. Foster City, Calif.) using the following thermal cycling profile: one cycle at 94°C for 5 min, followed by 30 cycles at 94°C for 30 s, at 64°C for 30 s, and at 72°C for 45 s, with a final extension step at 72°C for 10 min. After thermal cycling, 5 μl of each amplified product was separated by electrophoresis on a 1% agarose gel, stained with ethidium bromide, and visualized with UV light.

    PCR products were purified using a QIAquick PCR purification kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Then the PCR products were sequenced directly on an ABI PRISM 310 genetic analyzer using a Big Dye terminator cycle sequencing ready reaction kit (Applied Biosystems Japan Co. Ltd., Tokyo, Japan) as recommended by the kit manufacturers.

    Simultaneous detection of NIV and NL amplicons by PCR. We tested the suitability of our PCR protocol for the individual amplification of each DNA fragment, NIV (206 bp) and NL (650 bp) (5). For multiplex PCRs, a 5-μl aliquot of the DNA suspension was added to 45 μl of the PCR mixture described above, except that 20 pmol of each NL primer and 10 pmol of each NIV primer were used. The NL primers were used as an internal control to identify all species of fungus, while NIV primers were used to specifically detect C. nivariensis. In order to reduce the formation of nonspecific extension products, the protocol included a hot-start DNA amplification which was carried out using the following thermal cycling profile: one cycle at 94°C for 5 min, followed by 10 cycles at 94°C for 30 s, at 63°C for 20 s, and at 72°C for 15 s, and another 25 cycles at 94°C for 30 s, at 53.7°C for 20 s, and at 72°C for 20 s, culminating with a final extension step of 3 min.

    Using the newly designed primers, we were able to amplify a 206-bp fragment, as expected, from the three strains of C. nivariensis. In contrast, we failed to amplify the genomes from the list of unrelated microorganisms listed in Table 1.

    The ITS sequences of the three strains of C. nivariensis revealed that they do not have intraspecies variation, although further studies including new strains whenever detected will display a more reliable variation measure. However, the interspecies variation of C. nivariensis with other Candida species is remarkable. Indeed, the assay based on our newly designed primer set was optimized to yield the expected band for C. nivariensis but not for any of the other species examined.

    The species-specific primers for C. nivariensis presented here provide a molecular diagnostic method that can be used, in conjunction with current clinical tools, for the diagnosis of C. nivariensis infections with greater confidence and accuracy.

    Once the specific PCR for C. nivariensis was optimized, we approached the development of a multiplex PCR assay for detection of all fungal species and specific identification of C. nivariensis. In this respect, we performed a double amplification of the D1/D2 (large subunit rRNA gene) and NIV fragments. Figure 1 shows an agarose gel illustrating typical results obtained with the optimized multiplex PCR assay.

    Amplification of the D1/D2 and NIV targets produced easily identifiable bands consistent with their respective molecular sizes (650 and 206 bp, respectively). The NIV fragments were always amplified in the case of C. nivariensis strains but not in the case of infections by other Candida spp. The D1/D2 fragment was detected in all yeast strains.

    To understand the clinical significance and epidemiological role of C. nivariensis, it is very important to correctly identify this yeast in clinical specimens. The method reported is a very reliable assay for this purpose.

    ACKNOWLEDGMENTS

    This research was supported by Project BIO 2002/00953 from the Ministerio de Educacion y Ciencia (Spain) to S.M.-A. (partially supported by FIS contract 99/3060) and PI62/02 from the Fundacion Canaria de Investigacion y Salud (FUNCIS). S.M.-A. is an Associated Scientist of the Centro de Investigaciones Biologicas (CIB), Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.

    REFERENCES

    Alcoba-Florez, J., S. Mendez-álvarez, J. Cano, J. Guarro, E. Perez-Roth, and M. P. Arevalo. 2005. Phenotypic and molecular characterization of Candida nivariensis sp. nov., a possible new opportunistic fungus. J. Clin. Microbiol. 43:4107-4111.

    Chen, Y. C., J. D. Eisner, and M. M. Kattar. 2000. Identification of medically important yeasts using PCR-based detection of DNA sequence polymorphisms in the internal transcribed spacer 2 region of the rRNA genes. J. Clin. Microbiol. 38:2303-2310.

    Kurtzman, C. P., and H. J. Phaff. 1987. Molecular taxonomy, p. 63-94. In A. H. Rose and J. S. Harrison (ed.), The yeasts, vol. 1: biology of yeast. Academic Press, London, England.

    Kurtzman, C. P., and C. J. Robnett. 1997. Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA gene. J. Clin. Microbiol. 35:1216-1223.

    Kurtzman, C. P., and J. W. Fell. 1998. Summary of species characteristics, p.915-947. In C. P. Kurtzman and J. W. Fell (ed.), The yeasts, a taxonomic study, 4th ed. Elsevier Science B. V., Amsterdam, The Netherlands.

    Louws, F. J., J. L. W. Rademaker, and F. J. de Bruijin. 1999. The three Ds of PCR-based genomic analysis of phytobacteria: diversity, detection, and disease diagnosis. Annu. Rev. Phytopathol. 37:81-125.

    Maaroufi, Y., N. Ahariz, M. Husson, and F. Crokaert. 2004. Comparison of different methods of isolation of DNA of commonly encountered Candida species and its quantitation by using a real-time PCR-based assay. J. Clin. Microbiol. 42:3159-3163.

    Marr, K. A., and R. A. Bowden. 1999. Fungal infections in patients undergoing blood and marrow transplantation. Transpl. Infect. Dis. 1:237-246.

    Price, C. W., G. B. Fuson, and H. J. Phaff. 1978. Genome comparison in yeast systematics: delimitation of species within the genera Schwanniomyces, Saccharomyces, Debaryomyces, and Pichia. Microbiol. Rev. 42:161-193.

    Shin, J. H., F. S. Nolte, and C. J. Morrison. 1997. Rapid identification of Candida species in blood cultures by a clinically useful PCR method. J. Clin. Microbiol. 35:1454-1459.

    Shin, J. H., F. S. Nolte, B. P. Holloway, and C. J. Morrison. 1999. Rapid identification of up to three Candida species in a single reaction tube by a 5' exonuclease assay using fluorescent DNA probes. J. Clin. Microbiol. 37:165-170.

    Trama, J. P., E. Mordechai, and M. E. Adelson. 2005. Detection and identification of Candida vaginitis by real-time PCR and pyrosequencing. Mol. Cell. Probes 19:145-152.

日期:2007年5月10日 - 来自[2005年第43卷第12期]栏目

Results from the ARTEMIS DISK Global Antifungal Surveillance Study: a 6.5-Year Analysis of Susceptibilities of Candida and Other Yeast Species to Fluconazole

    University of Iowa College of Medicine, Iowa City, Iowa
    University of Texas Health Science Center, San Antonio, Texas
    University of Wales College of Medicine, Cardiff, United Kingdom
    Zhong Shan Hospital, Shanghai, China
    Institute of Antimicrobial Chemotherapy, Smolensk, Russia
    Hospital de Clinicas "Jose de San Martin," Buenos Aires, Argentina
    Institute of Clinical Microbiology, Faculty of Medicine, University of Szeged, Szeged, Hungary
    Giles Scientific, Inc., Santa Barbara, California

    ABSTRACT

    Fluconazole in vitro susceptibility test results for 140,767 yeasts were collected from 127 participating investigators in 39 countries from June 1997 through December 2003. Data were collected on 79,343 yeast isolates tested with voriconazole from 2001 through 2003. All investigators tested clinical yeast isolates by the CLSI (formerly NCCLS) M44-A disk diffusion method. Test plates were automatically read and results were recorded with the BIOMIC Vision Image Analysis System. Species, drug, zone diameter, susceptibility category, and quality control results were collected quarterly via e-mail for analysis. Duplicate (the same patient, same species, and same susceptible-resistant biotype profile during any 7-day period) and uncontrolled test results were not analyzed. The 10 most common species of yeasts all showed less resistance to voriconazole than to fluconazole. Candida krusei showed the largest difference, with over 70% resistance to fluconazole and less than 8% to voriconazole. All species of yeasts tested were more susceptible to voriconazole than to fluconazole, assuming proposed interpretive breakpoints of 17 mm (susceptible) and 13 mm (resistant) for voriconazole. MICs reported in this study were determined from the zone diameter in millimeters from the continuous agar gradient around each disk, which was calibrated with MICs determined from the standard CLSI M27-A2 broth dilution method by balanced-weight regression analysis. The results from this investigation demonstrate the broad spectrum of the azoles for most of the opportunistic yeast pathogens but also highlight several areas where resistance may be progressing and/or where previously rare species may be "emerging."

    INTRODUCTION

    Antifungal resistance surveillance with a focus on Candida is now widespread (5, 10, 17, 20, 29, 32). Most of these surveillance efforts are by necessity limited in terms of the numbers of participating sentinel sites and isolates tested. Furthermore, none of the programs is extensive enough to provide temporal and geographic data concerning the occurrence and resistance profiles of the less common Candida species and other, noncandidal opportunistic yeasts (21).

    The ARTEMIS Global Antifungal Surveillance Program is among the most comprehensive and long-running fungal surveillance programs (6, 12, 17, 19, 22, 24, 25, 27). The ARTEMIS Program is made up of two components: (i) a broad international network of participating sites (127 sites in 39 countries), each of which performs Clinical and Laboratory Standards Institute (CLSI, formerly National Committee for Clinical and Laboratory Standards [NCCLS])-recommended disk diffusion testing (M44-A) (14) of fluconazole and voriconazole against consecutive yeast isolates from a variety of clinical sources (ARTEMIS DISK Surveillance Study) (6), and (ii) a central reference laboratory (University of Iowa, Iowa City), where CLSI-recommended broth microdilution (BMD) MIC and disk diffusion testing (M27-A2 and M44-A, respectively) (13, 14) is performed on blood and normally sterile-site isolates of Candida and other opportunistic yeasts and molds thatare referred according to protocol from the participating ARTEMIS study sites (19, 22, 24, 25, 27). As such, the ARTEMIS Program has been designed to address many of the potential limitations of resistance surveillance studies (7): (i) it is both longitudinal (1997 to present) and global (127 participating sites in 39 countries) in scope, (ii) it employs standardized antifungal susceptibility test methods (CLSI disk [M44-A] and BMD MIC [M27-A2]) (13, 14), (iii) both internal quality control (QC) performed in each participating laboratory and external quality assurance measures are used to validate test results (25, 27), (iv) results are recorded electronically using the BIOMIC image analysis plate reader system (Giles Scientific, Santa Barbara, Calif.) (6, 19, 25, 27) and are stored in a central database, and (v) both Candida and non-Candida yeast isolates obtained from consecutive clinical samples from all body sites are tested locally, thus avoiding misleading results based on biased selective testing. This so-called "routine" testing is augmented by testing of isolates from blood and normally sterile sites in the central reference laboratory (25, 27). Thus, the ARTEMIS Program generates massive amounts of data that have been externally validated and that can be used to identify temporal and geographic trends in the species distribution of Candida and other opportunistic yeasts, as well as the resistance profiles of these organisms to fluconazole and voriconazole as determined by standardized CLSI disk diffusion testing.

    In the present study, we utilized the results from the ARTEMIS DISK Surveillance Program to evaluate global trends in the susceptibility of yeasts to fluconazole over a 6.5-year period (140,767 isolates from 127 study sites in 39 countries; June 1997 through December 2003). We also report results of voriconazole susceptibility testing performed on 79,343 isolates collected from 2001 to 2003. The scope of this study provides an unprecedented look at the occurrence and azole susceptibilities of several rare species of Candida, as well as several of the other opportunistic yeasts. The study is limited in that the numbers of isolates from certain regions are small and the time frame over which voriconazole data are available is relatively short.

    MATERIALS AND METHODS

    Organisms and test sites. A total of 134,715 isolates of Candida spp. and 6,052 isolates of noncandidal yeasts obtained from 127 different medical centers in Asia (23 sites), Latin America (16 sites), Europe (74 sites), the Middle East (2 sites), and North America (12 sites) were collected and tested against fluconazole between June 1997 and December 2003. In addition, a total of 79,343 isolates (75,810 isolates of Candida spp. and 3,533 other yeasts) from 115 study sites in 35 countries were tested against voriconazole between 2001 and 2003. All yeasts considered pathogens from all body sites (e.g., blood, normally sterile body fluids, deep tissue, genital tract, gastrointestinal tract, respiratory tract, skin, and soft tissue) and isolates from patients in all in-hospital locations during the study period were tested. Yeasts considered by the local site investigator to be colonizers, that is, not associated with an obvious pathology, were excluded, as were duplicate isolates from a given patient (the same species and the same susceptible-resistant biotype profile within any 7-day period). Identification of isolates was performed in accordance with each site's routine methods.

    Susceptibility test method. Disk diffusion testing of fluconazole and voriconazole was performed as described by Hazen et al. (6) and in CLSI document M44-A (14). Agar plates (150-mm diameter) containing Mueller-Hinton agar (obtained locally at all sites) supplemented with 2% glucose and 0.5 μg of methylene blue per ml (MH-MB) at a depth of 4.0 mm were used. The agar surface was inoculated by using a swab dipped in a cell suspension adjusted to the turbidity of a 0.5 McFarland standard. Fluconazole (25-μg) and voriconazole (1-μg) disks (Becton Dickinson, Sparks, Md.) were placed onto the surfaces of the plates, and the plates were incubated in air at 35 to 37°C and read at 18 to 24 h. Slowly growing isolates, primarily members of the genus Cryptococcus, were read after 48 h of incubation. Zone diameter endpoints were read at 80% growth inhibition by using the BIOMIC image analysis plate reader system (version 5.9; Giles Scientific, Santa Barbara, Calif.) (6, 19).

    The interpretive criteria for the fluconazole and voriconazole disk diffusion tests were those of the CLSI (1a,14): susceptible (S), zone diameters of 19 mm (fluconazole) and 17 mm (voriconazole); susceptible dose dependent (SDD), zone diameters of 15 to 18 mm (fluconazole) and 14 to 16 mm (voriconazole); and resistant (R), zone diameters of 14 mm (fluconazole) and 13 mm (voriconazole). The corresponding MIC breakpoints (13) are as follows: S, MIC of 8 μg/ml (fluconazole) and 1 μg/ml (voriconazole); SDD, MIC of 16 to 32 μg/ml (fluconazole) and 2 μg/ml (voriconazole); R, MIC of 64 μg/ml (fluconazole) and 4 μg/ml (voriconazole).

    QC. QC was performed in accordance with CLSI document M44-A (14) by using Candida albicans ATCC 90029 and C. parapsilosis ATCC 22019. A total of 5,865 and 5,484 QC results were obtained for fluconazole and voriconazole, respectively, of which more than 99% were within the acceptable limits.

    Analysis of results. All yeast disk test results were read by electronic image analysis and interpreted and recorded with a BIOMIC Plate Reader System (Giles Scientific Inc.). Test results were sent by e-mail to Giles Scientific for analysis. The zone diameter, susceptibility category (S, SDD, or R), and QC test results were all recorded electronically. In addition, MICs were calculated for each drug-organism pair by the BIOMIC System software. The MIC-versus-zone-diameter regression data used by the BIOMIC software were generated previously by ARTEMIS investigators (M.A.P. and M.G.R.) using CLSI BMD MIC and disk test methods (19, 25, 27). Patient and doctor names, duplicate test results (the same patient, the same species, and the same biotype results), and uncontrolled results were automatically eliminated by the BIOMIC system prior to analysis.

    RESULTS

    Isolation rates by species. A total of 140,767 yeast isolates were collected and tested at 127 study sites between June 1997 and December 2003 (Table 1). Candida species accounted for 95 to 97% of all isolates in each study year (overall, 95.7%). More than 16 different species of Candida were isolated, of which Candida albicans was the most common (overall, 66.2% of all Candida spp.). A decreasing trend in the rate of C. albicans isolation (overall decrease, 10 to 11%) was noted over the 6.5-year period. In contrast, increased rates of isolation of C. tropicalis (an increase of 2.9% from 1997 to 2003) and C. parapsilosis (an increase of 3.1% from 1997 to 2003) were noted. Neither C. glabrata nor C. krusei showed a consistent increase or decrease in isolation rate. Although isolates of more unusual Candida species, such as C. guilliermondii, C. kefyr, C. rugosa, and C. famata, constituted only a small percentage of the Candida isolates, the isolation rates of these four species increased from 2- to 10-fold over the course of the study. Likewise, although C. inconspicua, C. norvegensis, C. lipolytica, C. pelliculosa, and C. zeylanoides are rare species of Candida, the sheer size of the ARTEMIS database provides a significant number of each of these species for study.

    Among the noncandidal yeasts, Cryptococcus neoformans (21% of 6,052 isolates), Saccharomyces spp. (6.8%), Trichosporon spp. (6.5%), and Rhodotorula spp. (2.3%) were the most commonly identified species (Table 1). Unidentified ("other") yeasts represented 0.46 to 3.05% of all isolates. As noted previously (6), this percentage decreased somewhat over the course of the study as more isolates were identified to the species level.

    Fluconazole and voriconazole susceptibilities of Candida spp. Table 2 summarizes the in vitro susceptibilities of 78,463 and 75,787 isolates of Candida spp. to fluconazole and voriconazole, respectively, as determined by CLSI disk diffusion testing. These isolates were obtained from 115 institutions in 35 countries during the period 2001 through 2003. The distribution of zone diameters and their respective interpretive categories are shown in Fig. 1 for both agents. The percentages of isolates in each category (S, SDD, and R) were 89.6%, 4.0%, and 6.4% and 94.6%, 2.3%, and 3.1% for fluconazole and voriconazole, respectively. Fluconazole was most active against C. albicans (97.8% S), C. parapsilosis (93.2% S), C. lusitaniae (93.3% S), C. kefyr (95.3% S), C. dubliniensis (96.8% S), and C. pelliculosa (94.7% S). Decreased susceptibility to fluconazole was seen with C. glabrata (66.7% S; 16.6% R), C. krusei (9.4% S; 77.2% R), C. guilliermondii (73.3% S; 9.8% R), C. rugosa (39.3% S; 51.8% R), C. famata (79.8% S; 11.9% R), C. inconspicua (25.7% S; 49.2% R), C. norvegensis (50.0% S; 38.0% R), C. lipolytica (54.7% S; 39.6% R), and C. zeylanoides (54.1% S; 37.8% R). These findings confirm previously reported data for the more common species (e.g., C. albicans, C. glabrata, C. parapsilosis, and C. krusei) and markedly expand our understanding of the susceptibility, or lack thereof, of less common species, such as C. rugosa, C. inconspicua, and C. norvegensis, to fluconazole (5, 15, 16, 18, 21, 23).

    Voriconazole was significantly more active than fluconazole against virtually every species, with the exception of C. tropicalis (89.1% S to fluconazole versus 87.1% S to voriconazole) (Table 2). Among the species with decreased susceptibility to fluconazole, more than 80% were susceptible to voriconazole, including C. glabrata (81.7% S), C. krusei (83.2% S), C. guilliermondii (91.2% S), C. famata (89.5% S), C. inconspicua (89.2% S), and C. norvegensis (92.3% S). Among the fluconazole-resistant (zone diameter, 14 mm) isolates of C. glabrata, 30% remained susceptible (zone diameter, 17 mm) to voriconazole; however, all voriconazole-resistant strains were also resistant to fluconazole (reference 22 and data not shown). Although voriconazole was more active than fluconazole against C. rugosa (61.4% S versus 39.3% S, respectively), C. lipolytica (67.3% S versus 54.7% S, respectively), and C. zeylanoides (74.3% S versus 54.1% S, respectively), these species were markedly less susceptible and more resistant (11.4% to 26.4%) to voriconazole than all other species of Candida. Again, these data confirm and extend previous observations, especially with the less common species of Candida (18, 20, 23, 24). Importantly, it is readily apparent from these data that although some degree of cross-resistance may be seen between fluconazole and voriconazole, it varies by species and should not be assumed in the absence of species identification and susceptibility testing results.

    Trends in resistance to fluconazole among Candida spp. over a 6.5-year period. The longitudinal nature of the ARTEMISDISK Surveillance Program allows one to examine trends in fluconazole resistance among clinical isolates of Candida spp. with the important advantage of sufficient numbers of isolates of each species, all tested by a single standardized method (Table 3). Among the 10 species listed in Table 3, no consistent increase or decrease in fluconazole resistance was seen over time with C. albicans (range, 0.8% to 1.5%) or C. glabrata (range, 14.3% to 22.8%). Although resistance among C. tropicalis isolates appeared to decline from 1997-1998 (4.2%) thru 2001 (3.0%), increases were seen in 2002 (6.6%) and 2003 (5.0%). A slight increase in resistance was noted over time among C. parapsilosis and C. kefyr, whereas a major increase in resistance was detected among isolates of C. rugosa, where 61.2 to 66.0% resistance was observed in the last 2 years of data collection. In contrast, following a peak of 26.1% R in 2000, resistance among isolates of C. guilliermondii decreased steadily between 2001 (11.7% R) and 2003 (8.1% R). Although C. famata appeared to be quite resistant to fluconazole in 1997 and 1998 (47.4% of 19 isolates), this was likely due to the small number of isolates tested. As the numbers of C. famata isolates increased to >50 per year over the next 5 years, the level of resistance stabilized at 10 to 12%. Despite the increase in the overall percentage of isolates of C. krusei that tested as resistant to fluconazole, this is not an important finding, as the species must be considered to be clinically resistant to fluconazole. The CLSI recommends that C. krusei not be tested against fluconazole (13, 14). All such isolates should be reported as fluconazole resistant.

    Trends in resistance to voriconazole among Candida spp., 2001 to 2003. Voriconazole has been used clinically since 2001 and since that time has been tested against Candida in the ARTEMIS Global Surveillance Program (Table 4). Overall, there has been a slight increase in the percentage of Candida isolates that appear to be resistant (zone diameter, 13 mm) to voriconazole, from 2.6% in 2001 to 3.5% in 2003. This may be accounted for by increases in resistance observed with C. glabrata (9.8% to 11.0%), C. tropicalis (4.7% to 7.0%), C. rugosa (3.1 to 38.0%), C. lipolytica (7.7% to 12.0%), and unidentified Candida species (4.2% to 7.0%). In contrast, no change or a decrease in resistance was seen with C. albicans, C. parapsilosis, C. krusei, C. lusitaniae, C. kefyr, C. famata, C. inconspicua, C. dubliniensis, and C. pelliculosa. Thus, the picture for voriconazole, in terms of spectrum and potency versus Candida spp., looks quite favorable. Emerging resistance, especially among C. glabrata, C. tropicalis, and C. rugosa, bears close monitoring.

    Geographic variation in the susceptibilities of C. albicans and C. glabrata to fluconazole and voriconazole. Table 5presents the in vitro susceptibility results for fluconazole and voriconazole tested against the two most common species of Candida, C. albicans and C. glabrata, stratified by geographic region and country of origin for the time period 2001 to 2003. With the exception of those from India, isolates of C. albicans were highly susceptible to both fluconazole and voriconazole. The only other countries where the percentages of C. albicans susceptible to either agent dropped below 94% were Colombia (fluconazole, 91.2% S, 6.1% R) and Ecuador (fluconazole, 91.6% S, 4.9% R). Overall, there was no meaningful difference in the fluconazole or voriconazole susceptibility profile for C. albicans when stratified by specimen type (96.7% to 99.3% S to fluconazole; 97.9% to 99.3% S to voriconazole) or by hospital location (95.3 to 99.1% S to fluconazole; 97.2% to 99.4% S to voriconazole) (data not shown).

    Fluconazole and voriconazole susceptibilities of C. glabrata isolates varied considerably among the various countries and geographic regions. Susceptibilities to fluconazole were lowest (<50%) in Venezuela (29.2% S), Malaysia (34.0% S), Belgium (39.7% S), the Czech Republic (44.8% S), and South Africa (49.6%) and highest (>80%) in India and the Middle East (100% S), Brazil (94.9% S), Greece (93.9% S), Canada (90.6% S), Portugal (87.1% S), Mexico (86.7% S), Poland (86.4% S), South Korea (83.7% S), Turkey (82.4% S), and Italy (81.3% S). Overall rates of resistance to fluconazole among C. glabrata isolates were 10.6% in the Asia-Pacific region, 13.2% in Latin America, 16.5% in Europe, and 18.0% in North America (data not shown). These rates of fluconazole resistance are considerably higher for each geographic region than those reported previously for blood and normally sterile-site infection isolates of C. glabrata (range, 2 to 9% R) tested by BMD between 1992 and 2000 (20).

    In contrast to that seen with C. albicans, the susceptibility of C. glabrata isolates to fluconazole varied according to specimen type and hospital location. Isolates from blood and normally sterile sites were the most susceptible (71% S; 14.8% R) and genital tract isolates were the least susceptible (53.6% S; 21.2% R) to fluconazole (data not shown). The highest rates of resistance were seen in isolates of C. glabrata from the surgical intensive-care unit (21.3%), the obstetrics and gynecology service (21.5%), the hematology/oncology service (22.6%), and the neonatal intensive-care unit (35.0%) (data not shown).

    Voriconazole was equally or more active than fluconazole against C. glabrata isolates from all countries and geographic regions (Table 5). Susceptibilities to voriconazole were lowest (<70%) in Venezuela (32.7% S), Belgium (53.2% S), Malaysia (59.5% S), the Czech Republic (65.3% S), and Ecuador (66.7% S) and highest (>90%) in India, Turkey and the Middle East (100% S), Brazil (96.8% S), Canada (95.7% S), Greece (95.5%), Thailand (92.5%), and Portugal (90.0%). Overall rates of resistance to voriconazole among C. glabrata isolates were 4.1% in the Asia-Pacific region, 5.4% in Latin America, 5.6% in Europe, and 9.0% in North America (data not shown). Our previous results using BMD MIC testing found resistance rates of 2.2 to 5.4% among blood and normally sterile-site isolates of C. glabrata tested in 2001 and 2002 (22). Similar to that seen with C. albicans, there was little variation in the susceptibility of C. glabrata to voriconazole when stratified by specimen type. Isolates from blood and normally sterile sites were the most susceptible (81%) and genital tract isolates were the least susceptible (70%) to voriconazole (data not shown). The rates of resistance to voriconazole ranged from 2.5% (neonatal intensive-care unit) to 8.2% (hematology/oncology service) across the different hospital locations.

    Activities of fluconazole and voriconazole against other opportunistic yeasts and yeast-like fungi. Although they comprise only 3 to 5% of all of the isolates tested in this study, the number of noncandidal yeasts tested against fluconazole and voriconazole exceeds that published in the current literature (1, 3, 21, 26). Lack of standardized methods for testing most of these fungi may be considered problematic; however, the vast majority grew well on the MH-MB agar plates, and the zone diameters were easily determined. For the purposes of this study, we utilized the interpretive breakpoints for Candida, and we recognize that they may be adjusted for noncandidal yeasts in the future. Nevertheless, the data generated for these organisms are not dissimilar to those obtained using CLSI BMD MIC methods (1, 3, 21, 26). Using Cryptococcus neoformans as an example, the susceptibilities of the isolates shown in Table 6 indicated moderate susceptibility to fluconazole and a very high level of activity for voriconazole. Very similar findings for these two agents using BMD MIC methods were recently reported from our laboratory (26). As noted previously (21), most of these noncandidal yeasts were substantially less susceptible to both fluconazole and voriconazole than Candida species. Although voriconazole was more active than fluconazole for each of these different genera, it is notable that less than 80% of Trichosporon beigelii/Trichosporon cutaneum, Trichosporon asahi, and Rhodotorula spp. were susceptible to either of these agents. The diverse array of opportunistic yeasts and yeast-like fungi and their variable susceptibilities to these azole antifungals emphasize the need for prompt identification of noncandidal yeasts from clinical material. The flexibility of the CLSI disk diffusion method may well be an advantage in assessing the antifungal susceptibilities of these "emerging" pathogens.

    Conversion of zone diameters to MICs. In addition to using image analysis technology to measure and record the zones of inhibition surrounding an antifungal disk, the BIOMIC system uses previously developed scatter plots and regression analysis to calculate MICs based on the relationship between the zone diameter and the MIC (Fig. 2). The data in Fig. 2 show the correlation between the MIC and the zone diameter for voriconazole with Candida spp. As seen previously with fluconazole (6, 19, 25), an excellent correlation was observed. Based on these data, the voriconazole MICs for Candida spp. were calculated and the data were compared to BMD MICs published previously (24) for the same species (Table 7). Although the numbers of isolates tested are considerably different in the two groups, it is readily apparent that the MIC50 and MIC90 values are very close for each species, as is the percent resistant. Thus, the large amount of qualitative disk diffusion data presented here can be converted to quantitative MIC data for purposes of comparing the activities of fluconazole and voriconazole for individual species (Fig. 3) or potentially for following trends across time. Additional work in this area is warranted.

    DISCUSSION

    The ARTEMIS Global Antifungal Surveillance Program is the largest and most comprehensive program of its kind and the only one to incorporate many of the features that arguably constitute an "ideal" resistance surveillance program (7-9, 11, 30). It is longitudinal and global, employs standardized methods used for "routine" testing in participating laboratories and for "reference" testing in a central reference laboratory, uses electronic data capture and storage in a central database, and conducts external validation of the data generated by participating laboratories. The current report from the ARTEMIS DISK Surveillance Study includes more than 140,000 opportunistic yeast isolates and is by far the largest and most geographically diverse study of antifungal susceptibility and resistance to date (5, 15, 16, 20, 28). Important findings regarding species distribution include a steady decrease in the isolation of C. albicans and an increase in the isolation of C. tropicalis and C. parapsilosis. Although they are still rare, it appears that C. rugosa, C. famata, C. inconspicua, and C. norvegensis may be "emerging" in recent years. Among the noncandidal yeasts, Cryptococcus neoformans, Saccharomyces, Trichosporon, and Rhodotorula species are prominent and may prove to be important due to their decreased susceptibilities to several antifungal agents (21).

    Despite the use of a standard protocol, it is recognized that any surveillance program based on susceptibility tests performed by the participating laboratories needs to include some measure of quality assurance, beyond simple QC testing, in order to provide an independent assessment of laboratory performance and validation of the results generated by the various laboratories (7, 9, 31). One approach to cross-validation that has been suggested is to use centralized testing with high-quality microbiology to confirm the trends in routine data obtained from participating sentinel sites (7-9, 11, 30). Comparison of results obtained for isolates tested in participating laboratories with results obtained for the same organisms tested in a central reference laboratory would accomplish this goal (8, 9). This approach has been used to validate and support the epidemiologic relevance of findings from antibacterial surveillance programs (9, 30). Most recently, we have used the same approach to validate fluconazole and voriconazole disk test results generated by laboratories participating in the ARTEMIS Program (25, 27). More than 2,900 isolates of Candida obtained from blood and normally sterile-site infections were tested against fluconazole and voriconazole by ARTEMIS participating laboratories (CLSI disk test) and by the central reference laboratory (CLSI disk and BMD MIC tests) (25, 27). Categorical agreement between the reference MIC results and the disk diffusion test results performed in the participant laboratories was 87.4% and 94.1% for fluconazole and voriconazole, respectively (Table 8). A similar level of agreement was seen when the disk test results obtained in the reference laboratory were compared with those from the participant laboratories (references 25 and 27 and data not shown). It was noted that participating laboratories tended to err on the side of calling isolates more resistant than the reference laboratory did; however, the numbers of major and very major discrepancies were quite small (Table 8). This external quality assurance data, coupled with excellent QC performance, ensures the generation of accurate and useful surveillance data in the ARTEMIS DISK Surveillance Program.

    The data reported here for the more common species of Candida (i.e., C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis) confirm most of the previously published data regarding their susceptibilities to fluconazole and voriconazole (5, 16, 20, 24). The activity of fluconazole remains high against C. albicans, C. parapsilosis, and C. tropicalis, although resistance may be increasing among C. tropicalis isolates. Fluconazole resistance was considerable among isolates of C. glabrata, although the extent of resistance varied widely throughout the world. Fortunately, voriconazole remains quite active against this species. It is notable, however, that resistance to voriconazole has increased among C. glabrata isolates over the 3-year period of this study and was quite high in certain countries, such as Belgium (18.5%) and Venezuela (38.8%), where fluconazole resistance was also widespread. Again, our previous studies have shown that compared to reference laboratory testing of C. glabrata by MIC and disk methods, the fluconazole and voriconazole disk test results reported by ARTEMIS participating sites tended to overestimate resistance (25, 27). Thus, the rates of resistance to fluconazole and voriconazole reported in this study for C. glabrata may be somewhat higher than previously reported in the literature. Nevertheless, the geographical and temporal comparisons and differences remain important.

    The ARTEMIS database is most valuable as it pertains to the less common species of Candida (Table 2). The excellent activity of voriconazole against C. krusei was confirmed by the results from almost 2,000 clinical isolates. Similarly, the high levels of activity of both azoles against C. lusitaniae, C. kefyr, C. dubliniensis, and C. pelliculosa were clearly demonstrated, confirming previous results based on comparatively few isolates (21, 23). Equally important was the demonstration of generally poor activities of fluconazole against C. guilliermondii, C. rugosa, C. famata, C. inconspicua, C. norvegensis, C. lipolytica, and C. zeylanoides. In most instances, these findings confirm what can only be called preliminary observations (21); however, for some of these species, these constitute new data and serve to underscore the imperative to identify Candida to the species level. Although voriconazole is active against the vast majority of these rare species, it is notable that decreased susceptibility to this agent, as well as to fluconazole, is seen with C. rugosa, C. lipolytica, and C. zeylanoides. These findings are especially important for C. rugosa, as the frequency of isolation of this species appears to be increasing over time (Table 1), it has been shown to cause clusters of nosocomial infection that are poorly responsive to amphotericin B (2, 4), and it was previously considered highly susceptible to voriconazole based on results for less than 20 clinical isolates (21).

    As is the case for the less common Candida species, new information for noncandidal yeasts is provided by this data set. Although the antifungal susceptibility profile of Cryptococcus neoformans is well known (1, 26), much less is known of the susceptibilities of Saccharomyces, Trichosporon, Rhodotorula, and Blastoschizomyces species to fluconazole and voriconazole (3, 21, 33). The results presented in Table 6 indicate that most of these opportunistic yeasts have decreased susceptibility to fluconazole, and although voriconazole is clearly more active than fluconazole, decreased susceptibility to that agent is also seen with certain species of Trichosporon and with Rhodotorula spp. The fact that these yeast-like fungi are also nonsusceptible to the echinocandins (they lack -1,3-D-glucan) and respond variably to amphotericin B highlights the potential for their emergence as difficult-to-treat mycotic pathogens in the future (21, 33).

    Finally, the ability of the BIOMIC software to convert disk diffusion zone diameters to MICs is an important feature of the ARTEMIS surveillance program, providing quantitative data that will be valuable in trend analysis. We have extended the previous work of Hazen et al. (6) and have shown that the voriconazole MICs calculated from the disk diffusion data for Candida spp. compare very favorably to those obtained by BMD MIC testing performed centrally (Table 7).

    In summary, we present a tremendous volume of data describing temporal and geographic trends in the isolation and azole susceptibilities of opportunistic yeast pathogens. The data point to the strength of azole coverage for most of these organisms but also highlight several areas where resistance may be progressing and/or previously rare species may be "emerging." The strength of the ARTEMIS Global Surveillance Program is in the overall design, incorporating standardized test methods, "routine" and centralized testing of isolates, and a broad international network of study sites providing consistent data over time. The continued efforts of this surveillance program will provide data on pathogen frequency and antifungal susceptibility on a global scale.

    ACKNOWLEDGMENTS

    Linda Elliott provided excellent support in the preparation of the manuscript.

    The ARTEMIS DISK Surveillance Program is supported by grants from Pfizer.

    We express our appreciation to all ARTEMIS participants. Participants contributing to this study included Jorge Finquelievich, Buenos Aires University, and Nora Tiraboschi, Hospital Escuela Gral., Buenos Aires, Argentina; David Ellis, Women's and Children's Hospital, North Adelaide, Australia; Dominique Frameree, CHU de Jumet, Jumet, Annemarie van den Abeele, St Lucas Campus Heilige Familie, Ghent, and Jean-Marc Senterre, Hpital de la Citadelle, Liege, Belgium; Arnaldo Colombo, Escola Paulista de Medicina, Sao Paulo, Brazil; Robert Rennie, University of Alberta Hospital, Edmonton, and Steve Sanche, Royal University Hospital, Saskatoon, Canada; Bijie Hu, Zhong Shan Hospital, Shanghai, Yingchun Xu, Peking Union Medical College Hospital, Beijing, Yingyuan Zhang, Hua Shan Hospital, Shanghai, and Nan Shan Zhong, Guangzhou Institute of Respiratory Disease, Guangzhou, China; Pilar Rivas, Inst. Nacional de Cancerología, Bogota, Angela Restrepo and Catalina Bedout, CIB, Medellin, and Ricardo Vega and Matilde Mendez, Hospital Militar Central, Bogota, Colombia; Nada Mallatova, Hospital Ceske Budejovice, Ceske, and Eva Chmelarova, Krajska Hygienicka Stanice, Ostrava, Czech Republic; Julio Ayabaca, Hospital FF. AA HG1, Quito, and Jeannete Zurita, Hospital Vozandes, Quito, Ecuador; M. Mallie, Faculte de Pharmacie, Montpellier, and E. Candolfi, Institut de Parasitologie, Strasbourg, France; W. Fegeler, Universitaet Muenster, Münster, A. Haase, RWTH Aachen, Aachen, G. Rodloff, Inst. F. Med. Mikrobiologie, Leipzig, W. Bar, Carl-Thiem Klinikum, Cottbus, and V. Czaika, Humaine Kliniken, Bad Saarow, Germany; George Petrikos, Laikon General Hospital, Athens, Greece; Erzsebet Puskás, BAZ County Institute, Miskolc, Ilona Doczi, University of Szeged, Szeged, Mestyan Gyula, Medical University of Pecs, Pecs, and Radka Nikolova, Szt Laszlo Hospital, Budapest, Hungary; Uma Banerjee, All India Institute of Medical Sciences, New Delhi, India; Nathan Keller, Sheba Medical Center, Tel Hashomer, Israel; Vivian Tullio, Universita degli Studi di Torino, Turin, Gian Carlo Schito, University of Genoa, Genoa, Giacomo Fortina, Ospedale di Novara, Novara, Gian Piero Testore, Univerrsita di Roma Tor Vergata, Rome, Domenico D'Antonio, Pescara Civil Hospital, Pescara, Giorgio Scalise, Instituto di Malattie Infettive, Ancona, Pietro Martino, Dept. di Biotechnologie, Rome, and Graziana Manno, Universita di Genova, Genova, Italy; Kee Peng, University Malaya, Kuala Lumpur, Malaysia; Celia Alpuche and Jose Santos, Hospital General de Mexico, Mexico City, Eduardo Rodriguez Noriega, Universidad de Guadalajara, Guadalajara, andMussaret Zaidi, Hospital General O'Horan, Merida, Mexico; Jacques F. G. M. Meis, Canisius Wilhemina Hospital, Nijmegen, The Netherlands; Egil Lingaas, Rikshospitalet, Oslo, Norway; Danuta Dzierzanowska, Children's Memorial Health Institute, Warsaw, and Waclaw Pawliszyn, Pracownia Bakteriologii, Cracow, Poland; Mariada Luz Martins, Inst. de Higiene e Medicina Tropical, Lisbon, Luis Albuquerque, Centro Hospitalar de Coimbra, Coimbra, Laura Rosado, Instituto Nacional de Saude, Lisbon, Rosa Velho, Hosp. da Universidade de Coimbra, Coimbra, and Jose Amorim, Hospital de Santo Antonio, Porto, Portugal; Vera N. Ilina, Novosibirsk Regional Hospital, Novosibirsk, Olga I. Kretchikova, Institute of Antimicrobial Chemotherapy, Smolensk, Galina A. Klyasova, Hematology Research Center, Moscow, Sophia M. Rozanova, City Clinical Hospital No 40, Ekaterinburg, Irina G. Multykh, Territory Center of Laboratory Diagnostics, Krasnodar, Nikolay N. Klimko, Medical Mycology Research Institute, St. Petersburg, Elena D. Agapova, Irkutsk Regional Children's Hospital, Irkutsk, and Natalya V. Dmitrieva, Oncology Research Center, Moscow, Russia; Abdul Mohsen Al-Rasheed, Riyadh Armed Forces Hospital, Riyadh, Saudi Arabia; Jan Trupl, National Cancer Center, Leon Langsadl, NUTaRCH, Alena Vaculikova, Derer University Hospital, and Hupkova Helena, St. Cyril and Metod Hospital, Bratislava, Slovak Republic; Denise Roditi, Groote Schuur Hospital, Cape Town, Anwar Hoosen, GaRankuwa Hospital, Medunsa, H. H. Crewe-Brown, Baragwanath Hospital, Johannesburg, M. N. Janse van Rensburg, Pelanomi Hospital, UOFS, Bloemfontein, and Adriano Duse, Johannesburg General Hospital, Johannesburg, South Africa; Kyungwon Lee, Yonsei University College of Medicine, and Mi-Na Kim, Asan Medical Center, Seoul, South Korea; A. del Palacio, Hospital 12 De Octobre, and Aurora Sanchez-Sousa, Hospital Ramon y Cajal, Madrid, Spain; Jacques Bille, Institute of Microbiology CHUV, Lausanne, and K. Muhlethaler, Universitat Bern, Bern, Switzerland; Shan-Chwen Chang, National Taiwan University Hospital, Taipei, and Jen-Hsien Wang, China Medical College Hospital, Taichung, Taiwan; Malai Vorachit, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; Deniz Gur, Hacettepe University Children's Hospital, Ankara, and Volkan Korten, Marmara Medical School Hospital, Istanbul, Turkey; John Paul, Royal Sussex County Hospital, Brighton, Brian Jones, Glasgow Royal Infirmary, Glasgow, F. Kate Gould, Freeman Hospital, Newcastle, Chris Kibbler, Royal Free Hospital, London, Nigel Weightman, Friarage Hospital, Northallerton, Ian M. Gould, Aberdeen Royal Hospital, Aberdeen, Ruth Ashbee, General Infirmary, P.H.L. S, Leeds, and Rosemarie Barnes, University of Wales College of Medicine, Cardiff, United Kingdom; Jose Vazquez, Harper Hospital, Wayne State University, Detroit, Michigan; Ed Chan, Mt. Sinai Medical Center, New York, and Davise Larone, Cornell Medical Center NYPH, Ithaca, N.Y.; Ellen Jo Baron, Stanford Hospital and Clinics, Stanford, Calif.; Mahmoud A. Ghannoum, University Hospitals of Cleveland, Cleveland, Ohio; Mike Rinaldi, University of Texas Health Science Center, San Antonio, Texas; Kevin Hazen, University of Virginia Health Systems, Charlottesville, Va.; Elyse Foraker, Christiana Care, Wilmington, Del.; and Heidi Reyes, Gen del Este Domingo Luciani, and Axel Santiago, Universitario de Caracas, Caracas, Venezuela.

    REFERENCES

    Brandt, M. E., M. A. Pfaller, R. A. Hajjeh, R. H. Hamill, P. G. Pappas, A. L. Reingold, D. Rimland, and D. W. Warnock for the Cryptococcal Disease Active Surveillance Group. 2001. Trends in antifungal drug susceptibility of Cryptococcus neoformans isolates in the United States: 1992 to 1994 and 1996 to 1998.Antimicrob. Agents Chemother. 45:3065-3069.

    CLSI.2005 . Minutes of the CLSI Antifungal Subcommittee Meeting, 2005. CLSI, Wayne, Pa.

    Colombo, A. L., S. A. Melo, R. F. C. Rosas, R. Salomao, M. Briones, R. J. Hollis, S. A. Messer, and M. A. Pfaller. 2003. Outbreak of Candida rugosa candidemia: an emerging pathogen that may be refractory to amphotericin B therapy. Diagn. Microbiol. Infect. Dis. 46:253-257.

    Diekema, D. J., B. Petroelje, S. A. Messer, R. J. Hollis, and M. A. Pfaller. 2005. Activities of available and investigational antifungal agents against Rhodotorula species. J. Clin. Microbiol. 43:476-478.

    Dube, M. P., P. N. R. Heseltine, M. G. Rinaldi, S. Evans, and B. Zawacki. 1994. Fungemia and colonization with nystatin-resistant Candida rugosa in a burn unit. Clin. Infect. Dis. 18:77-82.

    Hajjeh, R. A., A. N. Sofair, L. H. Harrison, G. M. Lyon, B. A. Arthington-Skaggs, S. A. Mirza, M. Phelan, J. Morgan, W. Lee-Yang, M. A. Ciblak, L. E. Benjamin, L. Thompson Sanza, S. Huie, S. F. Yeo, M. E. Brandt, and D. W. Warnock.2004 . Incidence of bloodstream infections due to Candida species and in vitro susceptibilities of isolates collected from 1998 to 2000 in a population-based active surveillance program. J. Clin. Microbiol. 42:1519-1527.

    Hazen, K. C., E. J. Baron, A. L. Colombo, C. Girmenia, A. Sanchez-Sousa, A. del Palacio, C. de Bedout, D. L. Gibbs, and The Global Antifungal Surveillance Group.2003 . Comparison of the susceptibilities of Candida spp. to fluconazole and voriconazole in a 4-year global evaluation using disk diffusion. J. Clin. Microbiol. 41:5623-5632.

    Kahlmeter, G., and D. F. J. Brown. 2002. Resistance surveillance studies—comparability of results and quality assurance of methods. J. Antimicrob. Chemother. 50:775-777.

    Livermore, D. M., E. J. Threlfall, M. H. Reacher, A. P. Johnson, D. James, T. Cheasty, A. Shah, F. Warburton, A. V. Swan, J. Skinner, A. Grahm, and D. C. E. Speller. 2000. Are routine test data suitable for the surveillance of resistance Resistance rates amongst Escherchia coli from blood and CSF from 1991-1997, as assessed by routine and centralized testing. J. Antimicrob. Chemother. 45:205-211.

    Livermore, D. M., A. P. Macgowan, and M. C. J. Wale. 2005. Surveillance of antimicrobial resistance: centralized surveys to validate routine data offer a practical approach. Br. Med. J. 317:614-615.

    Luzzati, R., G. Amalfitano, L. Lazzarini, F. Soldori, S. Bellino, M. Solbiatic, M. C. Danzi, S. Vento, G. Todeschini, C. Vivenza, and E. Concia. 2000. Nosocomial candidemia in non-neutrophenic patients at an Italian tertiary care hospital.Eur. J. Clin. Microbiol. Infect. Dis. 19:602-607.

    Magee, J. T., M. L. Heginbothom, and B. W. Mason. 2005. Finding a strategy: the case for co-operative research on resistance epidemiology. J. Antimicrob. Chemother. 55:628-633.

    Meis, J., M. Petrou, J. Bille, D. Ellis, D. Gibbs, and the Global Antifungal Surveillance Group. 2000. A global evaluation of the susceptibility of Candida species to fluconazole by disk diffusion. Diagn. Microbiol. Infect. Dis. 36:215-223.

    National Committee for Clinical Laboratory Standards. 2002. Reference method for broth dilution testing of yeasts. Approved standard, 2nd ed. M27-A2. National Committee for Clinical Laboratory Standards, Wayne, Pa.

    National Committee for Clinical Laboratory Standards. 2004. Method for antifungal disk diffusion susceptibility testing of yeasts: approved guidance M44-A. National Committee for Clinical Laboratory Standards, Wayne, Pa.

    Nguyen, M. H., J. E. Peacock, Jr., A. J. Morris, D. C. Tanner, M. L. Nguyen, D. R. Snydman, M. M. Wagener, M. G. Rinaldi, and V. L. Yu. 1996. The changing face of candidemia: emergence of non-Candida albicans species and antifungal resistance. Am. J. Med. 100:617-623.

    Ostrosky-Zeichner, L., J. H. Rex, P. G. Pappas, R. J. Hamill, R. A. Larsen, H. W. Horowitz, W. G. Powderly, N. Hyslop, C. A. Kauffman, J. Cleary, J. E. Mangino, and J. Lee. 2003. Antifungal susceptibility survey of 2,000 bloodstream Candida isolates in the United States. Antimicrob. Agents Chemother. 47:3149-3154.

    Pfaller, M. A., and D. J. Diekema. 2002. Role of sentinel surveillance of candidemia: trends in species distribution and antifungal susceptibility. J. Clin. Microbiol. 40:3551-3557.

    Pfaller, M. A., D. J. Diekema, S. A. Messer, L. Boyken, R. J. Hollis, R. N. Jones, and the International Fungal Surveillance Participant Group.2003 . In vitro activities of voriconazole, posaconazole, and four licensed systemic antifungal agents against Candida species infrequently isolated from blood. J. Clin. Microbiol. 41:28-83.

    Pfaller, M. A., D. J. Diekema, S. A. Messer, L. Boyken, and R. J. Hollis. 2003. Activites of fluconazole and voriconazole against 1,586 recent clinical isolates of Candida species determined by broth microdilution, disk diffusion, and Etest methods: report from the ARTEMIS Global Antifungal Susceptibility Program, 2001. J. Clin. Microbiol. 41:1440-1446.

    Pfaller, M. A., and D. J. Diekema. 2004. Twelve years of fluconazole in clinical practice: global trends in species distribution and fluconazole susceptibility of bloodstream isolates of Candida. Clin. Microbiol. Infect. 10(Suppl. 1):11-23.

    Pfaller, M. A., and D. J. Diekema. 2004. Rare and emerging opportunistic fungal pathogens: concern for resistance beyond Candida albicans and Aspergillus fumigatus. J. Clin. Microbiol. 42:4419-4431.

    Pfaller, M. A., S. A. Messer, L. Boyken, S. Tendolkar, R. J. Hollis, and D. J. Diekema.2004 . Geographic variation in the susceptibilities of invasive isolates of Candida glabrata to seven systemically active antifungal agents: a global assessment from the ARTEMIS Antifungal Surveillance Program conducted in 2001 and 2002.J. Clin. Microbiol. 42:3142-3146.

    Pfaller, M. A., D. J. Diekema, S. A. Messer, L. Boyken, R. J. Hollis, and R. N. Jones.2004 . In vitro susceptibilities of rare Candida bloodstream isolates to ravuconazole and three comparative antifungal agents. Diagn. Microbiol. Infect. Dis. 48:101-105.

    Pfaller, M. A., S. A. Messer, L. Boyken, R. J. Hollis, C. Rice, S. Tendolkar, and D. J. Diekema.2004 . In vitro activities of voriconazole, posaconazole, and fluconazole against 4,169 clinical isolates of Candida spp. and Cryptococcus neoformans collected during 2001 and 2002 in the ARTEMIS global antifungal surveillance program.Diagn. Microbiol. Infect. Dis. 48:201-205.

    Pfaller, M. A., K. C. Hazen, S. A. Messer, L. Boyken, S. Tendolkar, R. J. Hollis, and D. J. Diekema. 2004. Comparison of results of fluconazole disk diffusion testing for Candida species with results from a central reference laboratory in the ARTEMIS Global Antifungal Surveillance Program. J. Clin. Microbiol. 42:3607-3612.

    Pfaller, M. A., S. A. Messer, L. Boyken, C. Rice, S. Tendolkar, R. J. Hollis, G. V. Doern, and D. J. Diekema. 2005. Global trends in the antifungal susceptibility of Cryptococcus neoformans (1990-2004). J. Clin. Microbiol. 43:2163-2167.

    Pfaller, M. A., L. Boyken, S. A. Messer, S. Tendolkar, R. J. Hollis, and D. J. Diekema.2005 . Comparison of results of voriconazole disk diffusion testing for Candida species with results from a central reference laboratory in the ARTEMIS Global Antifungal Surveillance Program. J. Clin. Microbiol. 43:5208-5213.

    Rees, J. R., R. W. Pinner, R. A. Hajjeh, M. R. Brandt, and A. L. Reingold.1998 . The epidemiological features of invasive mycotic infection in the San Francisco Bay Area, 1992-1993: results of a population-based laboratory active surveillance. Clin. Infect. Dis. 27:1138-1147.

    Richet, H., P. Roux, C. Des Champs, Y. Esnault, A. Andremont and the French Candidemia Study Group. 2002. Candidemia in French hospitals: incidence rates and characteristics. Clin. Microbiol. Infect. 8:405-412.

    Stelling, J. M., K. Travers, R. N. Jones, P. J. Turner, T. F. O'Brien, and S. B. Levy.2005 . Integrating Escherichia coli antimicrobiol susceptibility data from multiple surveillance programs. Emerg. Infect. Dis. 11:873-882.

    Tenover, F. C., M. J. Mohammed, J. Stelling, T. O'Brien, and R. Williams. 2001. Ability of laboratories to detect emerging antimicrobial resistance: proficiency testing and quality control results from the World Health Organizations' external quality assurance system for antimicrobial susceptibility testing.J. Clin. Microbiol. 39:241-250.

    Tortorano, A. M., J. Peman, H. Bernhardt, L. Klingspor, C. C. Kibbler, O. Faure, E. Biraghi, E. Canton, K. Zimmerman, S. Seaton, R. Grillot, and the ECMM Working Group on Candidaemia.2004 . Epidemiology of candidaemia in Europe: results of 28-month European Confederation of Medical Mycology (ECMM) hospital-based surveillance study. Eur. J. Clin. Microbiol. Infect. Dis. 23:317-322.

    Walsh, T. J., A. Groll, J. Hiemenz, R. Flemming, E. Roilides, and E. Anaissie. 2004. Infections due to emerging and uncommon medically important fungal pathogens. Clin. Microbiol. Infect. 10(Suppl. 1):48-66.

日期:2007年5月10日 - 来自[2005年第43卷第12期]栏目
循环ads

Use of Denaturing High-Performance Liquid Chromatography for Rapid Detection and Identification of Seven Candida Species

    Institute of Microbiology and Hygiene, Charite Universitaetsmedizin Berlin, Campus Charite Mitte, Dorotheenstrae 96, D-10117 Berlin, Germany
    Transgenomic Ltd., Omaha, Nebraska

    ABSTRACT

    A novel denaturing high-performance liquid chromatography (DHPLC)-based technique allows rapid high-resolution analysis of PCR products. We used this technique for unequivocal molecular identification of seven Candida species. We show the application of this PCR/DHPLC approach for direct detection and identification of yeast species from blood cultures and for detection of Candida colonization in the gastrointestinal tract of allogeneic transplant patients.

    INTRODUCTION

    The incidence of Candida infections has increased significantly over the past decades, being an important cause of morbidity and mortality in both critically ill and immunocompromised patients (1). Candida species colonize mucosal surfaces in humans and represent a major source for local and systemic endogenous infections. In addition, the ability of these yeasts to adhere to inert plastic surfaces (11) renders them important pathogens associated with indwelling catheters. Although Candida albicans is still the most prevalent species in clinical samples, the increasing incidence of non-C. albicans Candida species such as C. dubliniensis, C. krusei, C. glabrata, C. parapsilosis, C. tropicalis, C. guilliermondii, and C. lusitaniae has been reported (10, 13, 15, 19). Given the high lethality of invasive Candida infections, and due to various in vitro susceptibilities of different Candida species (10), rapid and reliable identification at the species level is required for correct treatment of these patients. Routine morphological and biochemical identification is time-consuming and laborious. Therefore, a number of PCR-based techniques have been developed for identification of medically important yeast species. The most popular targets for PCR amplification are the internal transcribed spacer regions ITS1 and ITS2 (2, 3, 6, 8, 16, 17). Methods used for identification of amplicons include hybridization with species-specific probes, direct sequencing, precise identification of the product length using a capillary sequencer, and characterization of restriction fragment length polymorphisms. However, most methods are inappropriate for the accurate analysis of clinical samples containing more than one yeast species. To overcome these limitations, we developed a novel technique based on PCR amplification of the variable ITS2 region and subsequent analysis of the amplicons by using the WAVE microbial analysis system (Transgenomic Ltd., Omaha, NE) (4, 5, 9, 18). This technique allows the analysis of complex samples containing more than one Candida species. Rapid verification of results can be done by direct sequencing of individual collected peaks.

    MATERIALS AND METHODS

    Yeast strains. We used reference strains from Candida membranaefaciens (ATCC 201377), Candida tropicalis (DSMZ 5991), Candida parapsilosis (ATCC 90018), Candida magnoliae (ATCC 201379), Candida lusitaniae (DSMZ 70102), Candida dubliniensis (CBS 7987), Candida albicans (ATCC 90028), Candida inconspicua (DSMZ 70631), Candida krusei (ATCC 6258), Cryptococcus neoformans (ATCC 90112), Candida kefyr (DSMZ 11954), Candida glabrata (ATCC 90030), Trichosporon mucoides (ATCC 201383), Candida norvegensis (DSMZ 70760), and Saccharomyces cerevisiae (ATCC 9763). In addition, clinical isolates of C. albicans (25 isolates), C. glabrata (22 isolates), C. tropicalis (9 isolates), C. kefyr (7 isolates), C. krusei (7 isolates), C. parapsilosis (7 isolates), and C. dubliniensis (12 isolates) were included to study the reproducibility of the method. Strains were collected consecutively in the clinical mycology laboratory from April through July 2001 from inpatients of the Charite University Hospital. The collection of C. albicans and C. glabrata strains was discontinued once 25 and 22 isolates of these species were identified, respectively. Copy strains were excluded.

    Isolation of DNA. Total DNA was isolated by phenol-chloroform extraction from 1 g of feces, 200 μl of blood culture fluid, or 200 μl of aqueous suspensions of yeast strains according to modified protocols (20). Samples were homogenized in 1 ml lysis buffer (500 mM Tris, pH 9.0, 20 mM EDTA, 10 mM NaCl, 1% sodium dodecyl sulfate) with 10 μl proteinase K (10 U/ml) and incubated at 56°C. After 1 h, 150 μl of phenol and 0.2 g of zirconium beads (0.1 mm; BioSpec) were added, and cells were disintegrated by shaking three times for 30 s in a FastPrep FP120 (Bio101) device. The aqueous phase was transferred to a fresh sterile tube, and DNA was further purified by chloroform/isoamyl alcohol (24:1) extraction. After centrifugation at 16,000 x g for 5 min, the aqueous phase was transferred to a fresh sterile tube, and DNA extraction was repeated with chloroform/isoamyl alcohol (24:1). The aqueous phase was transferred to a fresh sterile tube, and DNA was precipitated on ice with 0.1 volume of sodium acetate (3 M) and 2.5 volumes of 99% (vol/vol) ethanol. After an additional centrifugation at 16,000 x g for 20 min, the supernatant fluid was discarded. The DNA pellet was washed twice with 70% (vol/vol) ethanol, dried in a Speed Vac, and dissolved in 100 μl of Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8.0). Extraction of pure lysis buffer was performed in parallel as a negative control.

    Conditions for PCR amplification. ITS2 amplification primers (MycoSeq, catalog no. 79018/19) were obtained from Transgenomic (Omaha, NE). The PCR mixture (total volume, 50 μl) consisted of 1 μl of template, 0.5 μM of forward and reverse primers, 5 μl of 10x Optimase buffer (Transgenomic, Omaha, NE), 400 μM of each deoxyribonucleotide triphosphate (Roche Diagnostics, Mannheim, Germany), and 2.5 U of Optimase DNA polymerase (Transgenomic, Omaha, NE). PCR products were generated using a T3 thermocycler (Biometra, Goettingen, Germany) with the following touchdown parameters: a denaturation step at 95°C for 2 min and 14 cycles at 95°C for 30 s, 61.3°C for 30 s with a –0.5°C increment every 30 s (reaching 54.3°C after 14 cycles), and 72°C for 40 s, followed by 19 cycles at 95°C for 30 s, 54.3°C for 30 s, and 72°C for 40 s and a final extension step at 72°C for 5 min. PCR products were verified on 2% (wt/vol) agarose gel in 1x Tris-borate-EDTA buffer after ethidium bromide staining.

    DHPLC analysis. PCR products were analyzed by ion-pair reverse-phase denaturing high-performance liquid chromatography (DHPLC) on the WAVE microbial analysis system (Transgenomic, Omaha, NE). Basically, 1 μl of each PCR product was loaded onto the autosampler of the system. PCR products were separated on a DNASep HT column cartridge (catalog no. 993710; Transgenomic, Omaha, NE). Separation conditions were adapted by temperature, flow rate, and gradient. The optimal separation conditions are listed in Table 1. PCR products were stained by using an intercalating stain (HS staining solution I, catalog no. 553440; Transgenomic, Omaha, NE) and visualized with an HSX-3500 fluorescence detector. In some experiments, unstained PCR products were analyzed using a UV detector. All buffer solutions were obtained from Transgenomic (Omaha, NE). Results were analyzed using Navigator software version 1.5.4 (Build 23).

    Sequence analysis. ITS2 amplicons from selected peak fractions were collected with the FCW 200 DNA fragment collector. The volume of each fraction varied from 100 to 200 μl depending upon the peak size and height. An aliquot was applied as a template for reamplification by PCR using the same primers and conditions described above. Purified PCR products (PCR purification kit; QIAGEN, Hilden, Germany) served as a template for sequencing reactions using the chemicals and protocols from Beckman Coulter (Fullerton, CA). ITS2 sequences were determined with the CEQ8000 DNA sequencing system (Beckman Coulter, Fullerton, CA) as recommended by the vendor. The sequences were compared with entries in public databases by using the BLAST software (BlastN algorithm) provided by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov).

    Conditions for blood culture analysis. Growth-positive BacT/ALERT FA (bioMerieux) blood culture bottles were analyzed by Gram stain. Microscopically positive materials (yeast cells in Gram stain) were subcultured on Sabouraud agar and CHROMagar (Mast Diagnostics) and incubated at 30°C and 37°C, respectively. Yeasts grown for 2 to 7 days were subcultured and identified using phenotypical and molecular methods as described previously (7, 8, 12, 14, 16). For some experiments, blood culture bottles from our clinical diagnostic microbiology laboratory that did not show growth after 7 days of incubation were spiked with 2 x 105 yeast cells (final concentration, 5 x 103 yeast cells/ml) and incubated under routine conditions. When growth was indicated by the system, yeasts were identified as described above. Aliquots of each blood culture were used for DNA isolation and subsequent PCR/DHPLC analysis.

    Collection and treatment of fecal samples. Stool samples were taken from allogeneic stem cell recipients. Fresh fecal samples (2 to 3 g) were aliquoted, and part (0.5 to 1 g) was transferred to 1.5 ml nonselective liquid culture medium (10 g/liter tryptone, 5 g/liter yeast extract, 10 g/liter NaCl) containing 70% (vol/vol) glycerin and immediately frozen at –80°C for cultural analysis. For culture, aliquots of 100 μl each of the stool samples were plated onto Sabouraud agar and CHROMagar (Mast Diagnostics) and incubated at 30°C and 37°C, respectively. In addition, 4 ml Sabouraud medium was inoculated with 200 μl of the stool sample and incubated for 2 days at 30°C. One hundred microliters of this suspension was then spread on Sabouraud agar and CHROMagar (Mast Diagnostics) and incubated at 30°C and 37°C, respectively. Identification of yeasts was done using phenotypic tests as described above. Remaining portions (1 to 1.5 g) were transferred to sterile plastic tubes containing 10 ml lysis buffer for subsequent DNA extraction.

    RESULTS

    Universal fungus-specific primers flanking the ITS2 region complementary to conserved regions of the 5.8S and 28S rRNA genes, respectively, were used to generate PCR products ranging from 200 to 300 bp.

    The PCR/DHPLC approach with ITS2 as the target region was evaluated using reference strains from Candida membranaefaciens, Candida tropicalis, Candida parapsilosis, Candida magnoliae, Candida lusitaniae, Candida dubliniensis, Candida albicans, Candida inconspicua, Candida krusei, Cryptococcus neoformans, Candida kefyr, and Candida glabrata. Both single and mixed ITS2 amplicons were injected into the WAVE microbial analysis system (Fig. 1). Resulting peak profiles showed unique signals for each reference strain. Hence, a mixture of these PCR products was used as a marker for the identification of fungal DNA (Fig. 1). Reference strains from Trichosporon mucoides, Candida norvegensis, and Saccharomyces cerevisiae showed unique peak profiles but were excluded from the marker mixture to limit the complexity of the marker set (data not shown). Reproducibility was studied using clinical isolates of C. albicans (25 isolates), C. glabrata (22 isolates), C. tropicalis (9 isolates), C. kefyr (7 isolates), C. krusei (7 isolates), C. parapsilosis (7 isolates), and C. dubliniensis (12 isolates). Although major peaks were consistent with the marker, some additional side peaks were observed (Fig. 2). Thus, in addition to strains exhibiting one major peak only (type I), six variant profiles of C. albicans (types II to VII), five additional profiles of C. glabrata (types II to VI), and two additional profiles of C. tropicalis (types II and III) could be distinguished (Fig. 2).

    To simulate diagnostic procedures for candidemia, a total of 14 negative blood cultures was spiked in duplicate with pure cultures of reference strains of C. parapsilosis, C. tropicalis, C. albicans, C. glabrata, C. dubliniensis, C. krusei, and a mixture of C. albicans and C. dubliniensis. This panel of blood cultures was blinded for subsequent DHPLC analysis. Total DNA was extracted from positive blood cultures and PCR amplified. Resulting DHPLC peaks of the panel were assigned to the corresponding reference peaks of the Candida marker set. All Candida species tested were unequivocally identified. In a second set of experiments, the assay was applied to real clinical specimens. Positive blood cultures (yeast cells in Gram stain) consecutively collected in the clinical mycology laboratory of our institution from November 2004 through June 2005 were analyzed using the PCR/DHPLC approach. The 66 blood culture samples studied comprised 26 copy strains, e.g., the second or third isolates from independent blood samples of the same patient. Results were in agreement with those achieved by culture analysis. Most samples (34 out of 66 samples) were positive for C. albicans. Other samples contained C. dubliniensis (1 sample), C. parapsilosis (2 samples), C. glabrata (12 samples), C. krusei (8 samples), and C. tropicalis (7 samples). Moreover, in one blood culture, the WAVE system identified a mixed infection with C. albicans and C. glabrata. One Trichosporon asahii infection showed a major peak in DHPLC that could not be resolved since T. asahii was not included in the marker used as a reference for species identification. While identification of major peaks was simple by using the marker mixture as a reference, side peaks were observed in some samples. All profiles could be assigned to variants shown in Fig. 2. The distribution of DHPLC variants in the strains or samples studied is given in Table 2.

    In addition, this novel method was applied for detecting yeast populations in human feces to test its ability for future monitoring of fungal colonization of mucosal surfaces. Fecal samples from four hospitalized patients were tested by PCR/DHPLC, and results were similar to those obtained by cultural analysis. Two samples were positive for C. glabrata. Furthermore, in one stool sample, the WAVE system identified mixed colonization with C. albicans and C. kefyr. Again, this result was confirmed by culture analysis. One sample gave divergent molecular and cultural results. While C. albicans. was detected by both methods, colonization by C. glabrata was found by DHPLC only. In contrast, Geotrichum candidum was cultivated from this sample but not found by PCR/DHPLC.

    DISCUSSION

    Results demonstrate that the PCR/DHPLC method is suited to identify fungal species in clinical specimens, e.g., blood culture and fecal samples. All strains tested, mainly Candida spp., were characterized by unique peak profiles. Amplicons from 12 fungal strains were combined and used as a marker for the identification of yeasts in clinical sample material. Seventy-nine of a total of 80 blood culture samples studied (14 spiked and 66 clinical samples) were correctly identified. Side peaks observed in some cases did not affect the identification of the respective Candida species. Rare ambiguities could be resolved by collecting and PCR/sequencing of the respective DHPLC peak. The more complex profiles found in three species allowed the identification of subtypes, an observation that may be of importance for epidemiological studies. PCR/DHPLC variants were observed in C. albicans, C. glabrata, and C. tropicalis. In C. albicans and C. glabrata, the single-peak profile (type I) is much more prevalent than any PCR/DHPLC profile with side peaks (Table 2). While PCR/DHPLC subtypes of Candida spp. have not been analyzed in detail, we speculate that variants may occur due to microheterogeneities between various rRNA gene clusters of a genome.

    Taken together, we conclude that this method may replace the time-consuming and sometimes challenging culture-based identification of yeast species. Assuming that DNA extraction takes 4 hours, PCR takes 3 hours, and DHPLC on the WAVE system takes 30 minutes, results can be obtained in 1 working day. Processing of blood culture samples may be further accelerated by using a commercial genomic DNA blood kit.

    The applications of the WAVE microbial analysis system presented here capitalize on the ability of the system to unequivocally identify more than one yeast species in a given sample. Therefore, in addition to rapid and simple identification of yeasts from positive blood cultures, the system enables fast and reliable detection of yeasts in complex human microbiota, e.g., intestinal microflora or skin, and should be helpful for epidemiological studies.

    ACKNOWLEDGMENTS

    We thank Gernot Reifenberger for excellent technical assistance.

    This work was supported by the Deutsche Forschungsgemeinschaft (grant GO 363/10-1 to U.B.G.).

    O.G. and S.H. contributed equally to this work.

    REFERENCES

    Chen, Y. C., S. C. Chang, C. C. Sun, L. S. Yang, W. C. Hsieh, and K. T. Luh. 1997. Secular trends in the epidemiology of nosocomial fungal infections at a teaching hospital in Taiwan, 1981 to 1993. Infect. Control Hosp. Epidemiol. 18:369-375.

    Chen, Y. C., J. D. Eisner, M. M. Kattar, S. L. Rassoulian-Barrett, K. LaFe, U. Bui, A. P. Limaye, and B. T. Cookson. 2001. Polymorphic internal transcribed spacer region 1 DNA sequences identify medically important yeasts. J. Clin. Microbiol. 39:4042-4051.

    De Baere, T., G. Claeys, D. Swinne, G. Verschraegen, A. Muylaert, C. Massonet, and M. Vaneechoutte. 2002. Identification of cultured isolates of clinically important yeast species using fluorescent fragment length analysis of the amplified internally transcribed rRNA spacer 2 region (ITS2). BMC Microbiol. 2:21.

    Domann, E., G. Hong, C. Imirzalioglu, S. Turschner, J. Kuhle, C. Watzel, T. Hain, H. Hossain, and T. Chakraborty. 2003. Culture-independent identification of pathogenic bacteria and polymicrobial infections in the genitourinary tract of renal transplant recipients. J. Clin. Microbiol. 41:5500-5510.

    Frueh, F. W., and M. Noyer-Weidner. 2003. The use of denaturing high-performance liquid chromatography (DHPLC) for the analysis of genetic variations: impact for diagnostics and pharmacogenetics. Clin. Chem. Lab. Med. 41:452-461.

    Fujita, S., B. A. Lasker, T. J. Lott, E. Reiss, and C. J. Morrison. 1995. Microtitration plate enzyme immunoassay to detect PCR-amplified DNA from Candida species in blood. J. Clin. Microbiol. 33:962-967.

    Graf, B., T. Adam, E. Zill, and U. B. Gbel. 2000. Evaluation of the VITEK 2 system for rapid identification of yeasts and yeast-like organisms. J. Clin. Microbiol. 38:1782-1785.

    Graf, B., A. Trost, J. Eucker, U. B. Gbel, and T. Adam. 2004. Rapid and simple differentiation of C. dubliniensis from C. albicans. Diagn. Microbiol. Infect. Dis. 48:149-151.

    Hurtle, W., D. Shoemaker, E. Henchal, and D. Norwood. 2002. Denaturing HPLC for identifying bacteria. BioTechniques 33:386-388, 390-391.

    Kao, A. S., M. E. Brandt, W. R. Pruitt, L. A. Conn, B. A. Perkins, D. S. Stephens, W. S. Baughman, A. L. Reingold, G. A. Rothrock, M. A. Pfaller, R. W. Pinner, and R. A. Hajjeh. 1999. The epidemiology of candidemia in two United States cities: results of a population-based active surveillance. Clin. Infect. Dis. 29:1164-1170.

    Klotz, S. A., D. J. Drutz, and J. E. Zajic. 1985. Factors governing adherence of Candida species to plastic surfaces. Infect. Immun. 50:97-101.

    Kreger-Van Rij, N. J. W. 1984. The yeasts: a taxonomic study. Elsevier, Amsterdam, The Netherlands.

    Malani, P. N., S. F. Bradley, R. S. Little, and C. A. Kauffman. 2001. Trends in species causing fungaemia in a tertiary care medical centre over 12 years. Mycoses 44:446-449.

    Pinjon, E., D. Sullivan, I. Salkin, D. Shanley, and D. Coleman. 1998. Simple, inexpensive, reliable method for differentiation of Candida dubliniensis from Candida albicans. J. Clin. Microbiol. 36:2093-2095.

    Swinne, D., M. Watelle, C. Suetens, K. Mertens, P. A. Fonteyne, and N. Nolard. 2004. A one-year survey of candidemia in Belgium in 2002. Epidemiol. Infect. 132:1175-1180.

    Trost, A., B. Graf, J. Eucker, O. Sezer, K. Possinger, U. B. Gbel, and T. Adam. 2004. Identification of clinically relevant yeasts by PCR/RFLP. J. Microbiol. Methods 56:201-211.

    Turenne, C. Y., S. E. Sanche, D. J. Hoban, J. A. Karlowsky, and A. M. Kabani. 1999. Rapid identification of fungi by using the ITS2 genetic region and an automated fluorescent capillary electrophoresis system. J. Clin. Microbiol. 37:1846-1851.

    Xiao, W., and P. J. Oefner. 2001. Denaturing high-performance liquid chromatography: a review. Hum. Mutat. 17:439-474.

    Yang, C. W., T. M. Barkham, F. Y. Chan, and Y. Wang. 2003. Prevalence of Candida species, including Candida dubliniensis, in Singapore. J. Clin. Microbiol. 41:472-474.

    Zoetendal, E. G., A. D. Akkermans, and W. M. De Vos. 1998. Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples reveals stable and host-specific communities of active bacteria. Appl. Environ. Microbiol. 64:3854-3859.

日期:2007年5月10日 - 来自[2005年第43卷第12期]栏目

In Vitro Activities of Anidulafungin against More than 2,500 Clinical Isolates of Candida spp., Including 315 Isolates Resistant to Fluconazole

    Departments of Pathology Epidemiology
    Medicine, University of Iowa College of Medicine and College of Public Health, Iowa City, Iowa 52242

    ABSTRACT

    Anidulafungin is an echinocandin antifungal agent with potent activity against Candida spp. We assessed the in vitro activity of anidulafungin against 2,235 clinical isolates of Candida spp. using the CLSI broth microdilution method. Anidulafungin was very active against Candida spp. (the MIC at which 90% of strains are inhibited [MIC90] was 2 μg/ml when MIC endpoint criteria of partial inhibition [MIC-2] were used). Candida albicans, C. glabrata, C. tropicalis, C. krusei, and C. kefyr were the most susceptible species of Candida (MIC90, 0.06 to 0.12 μg/ml), and C. parapsilosis, C. lusitaniae, and C. guilliermondii were the least susceptible (MIC90, 0.5 to 2 μg/ml). In addition, 315 fluconazole-resistant isolates were tested, and 99% were inhibited by 1 μg/ml of anidulafungin. These results provide further evidence for the spectrum and potency of anidulafungin activity against a large and geographically diverse collection of clinically important isolates of Candida spp.

    INTRODUCTION

    Anidulafungin is an investigational echinocandin with potent fungicidal activity against many species of Candida (1, 4, 5, 6, 11, 12, 13, 14, 17, 18). Anidulafungin is now in phase III clinical trials and has been shown to be safe and efficacious in treating invasive candidiasis (8). Although several studies documenting the in vitro activity of anidulafungin against Candida spp. have been published, these studies employed test methods that utilize either a more conservative MIC endpoint criterion (100% inhibition or MIC-0), an extended incubation time (48 h), or both and are either limited in the number of isolates of the various species of Candida tested or are restricted in the geographical distribution of the tested strains (1, 4, 11, 14, 17, 18).

    In the present study, we determined the in vitro activity of anidulafungin against an international collection of more than 2,000 clinical isolates of Candida representing predominately bloodstream infection and other invasive forms of candidiasis. We also provide an evaluation of anidulafungin activity against 315 isolates with resistance (MIC, 64 μg/ml) to fluconazole. Because there is some controversy over the optimal method for performing in vitro susceptibility testing of the echinocandins (3), there is currently no standardized method for testing these agents against Candida. We have elected to use the method recommended by the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS), which employs RPMI 1640 broth, 24 h of incubation, and a partial inhibition (MIC-2, or 50% inhibition relative to control) MIC endpoint (3, 9, 10, 15, 16). The results are presented as the cumulative percentage of isolates inhibited at each concentration throughout the dilution series (full-range MICs) to facilitate comparison with other studies using the CLSI method.

    MATERIALS AND METHODS

    Organisms. A total of 2,235 clinical isolates of Candida spp. obtained from 91 different medical centers internationally were tested. These isolates were contributed as part of an ongoing prospective surveillance program and represent the incident isolate obtained from a given infectious episode. The collection included the following numbers of isolates: C. albicans, 1,181; C. glabrata, 265; C. parapsilosis, 328; C. tropicalis, 278; C. krusei, 59; C. lusitaniae, 34; C. guilliermondii, 57; C. kefyr, 15; and miscellaneous Candida spp., 18. The isolates represented the Asia-Pacific region (354 isolates from 16 study sites), Latin America (542 isolates from 15 study sites), Europe (668 isolates from 32 study sites), and North America (671 isolates from 28 study sites) (Table 1). In addition, a collection of 315 isolates of Candida spp. previously characterized as resistant to fluconazole (MIC, 64 μg/ml) (15) was tested in order to determine the activity of anidulafungin against these clinically important strains: C. albicans (41 isolates), C. glabrata (110 isolates), C. krusei (146 isolates), and Candida spp. (18 isolates). The isolates were all recent clinical isolates (2001 to 2004) and were from blood or normally sterile body fluids (cerebrospinal fluid, pleural fluid, or peritoneal fluid) and tissue. The isolates were identified by standard methods (7) and were stored as water suspensions until they were used in the study.

    Antifungal agents. Standard antifungal powder of anidulafungin (Vicuron, Inc., King of Prussia, PA) was obtained from the manufacturer. Stock solutions were prepared in dimethyl sulfoxide. Serial twofold dilutions were prepared exactly as outlined in CLSI document M27-A2 (9). Final dilutions were made in RPMI 1640 medium (Sigma, St. Louis, MO) buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) buffer (Sigma). Aliquots (0.1 ml) of the antifungal agent at twice the final concentration were dispensed into wells of plastic microdilution trays. The trays were sealed and frozen at –70°C until they were used.

    Antifungal susceptibility studies. Broth microdilution testing of all 2,550 isolates was performed as described previously (16) in accordance with the guidelines of CLSI document M27-A2 (9), using a final inoculum concentration of (1.5 ± 1.0) x 103 cells/ml, RPMI 1640 medium, and incubation at 35°C for 24 h. MIC endpoints for anidulafungin were defined as the lowest concentration that produced a prominent decrease in turbidity (50% or MIC-2) relative to that of the drug-free control well (2, 9, 16).

    RESULTS AND DISCUSSION

    The species distribution of the isolates tested, stratified by the geographic region of origin, is shown in Table 1. All of the major species were represented, including less common and "emerging" species, such as C. guilliermondii. It is notable that the species distributions of Candida bloodstream infection isolates contributed by study sites in the Asia-Pacific and Latin American regions were considerably different than that seen in North America.

    Whereas C. glabrata was much more frequently isolated than either C. parapsilosis or C. tropicalis in North America, it was less common than either of these two species in the Asia-Pacific region and in Latin America. Similarly, C. parapsilosis was more common than C. glabrata among isolates contributed from Europe. Among the less frequently isolated species of Candida, C. krusei was more common among European isolates and C. guilliermondii was more common in Latin America, where it ranked above both C. glabrata and C. krusei among all bloodstream infection isolates. The species diversity, the number of contributing study sites, and the broad (worldwide) geographic representation are strengths of this database.

    Table 2 summarizes the in vitro susceptibilities of 2,235 isolates of Candida spp. to anidulafungin using the MIC-2 endpoint method described above. Overall, anidulafungin was quite active against this broad range of Candida species (MIC at which 50% of the strains are inhibited [MIC50], 0.06 μg/ml; MIC90, 2 μg/ml). C. albicans, C. glabrata, C. tropicalis, C. krusei, and C. kefyr were the species most susceptible to anidulafungin (MIC90, 0.06 to 0.12 μg/ml), and C. parapsilosis (MIC90, 2 μg/ml), C. lusitaniae (MIC90, 0.5 μg/ml), and C. guilliermondii (MIC50, 2 μg/ml) were the least susceptible. Notably, 100% of C. glabrata and C. krusei isolates were inhibited by 0.25 μg/ml of anidulafungin. Despite differences in the species distribution across the four geographic regions, there was no difference in the activity of anidulafungin, overall or by species, when stratified by region.

    The activity of anidulafungin against the 315 fluconazole-resistant isolates (Table 3) was equal to or better than its activity against the larger group of more azole-susceptible strains (Table 2). Notably, 100% of fluconazole-resistant isolates of C. glabrata and C. krusei were inhibited by 0.5 μg/ml of anidulafungin (Table 3).

    These findings confirm and extend those reported previously regarding the anticandidal activity of anidulafungin (1, 4, 11, 15, 17, 18). Anidulafungin exhibited potent activity against virtually all species of Candida, including those with resistance to fluconazole. As seen with caspofungin (15, 16), there appear to be two broad groups of Candida species that can be differentiated by the degree of susceptibility to anidulafungin. One group includes the common species C. albicans, C. glabrata, C. tropicalis, and C. krusei (as well as the less common C. kefyr) and is highly susceptible to anidulafungin (MIC90, 0.12 μg/ml), whereas the second group includes C. parapsilosis and less common species, such as C. lusitaniae and C. guilliermondii, and is 4- to 16-fold less susceptible to anidulafungin (MIC90, 0.5 to 2 μg/ml) (Table 2). Preliminary data suggest that all of these species may respond clinically in a similar fashion to anidulafungin treatment (8). The MICs for 99% of isolates in both groups are 2 μg/ml when tested using the partial inhibition endpoint criteria, a concentration that is exceeded throughout the dosing interval following the administration of anidulafungin at standard doses of 100 mg/day (8, 13).

    In summary, we have demonstrated that anidulafungin has potent in vitro activity against a broad range of Candida species from throughout the world. The emerging in vivo data from animal models and from clinical trials support the efficacy of anidulafungin in the treatment of invasive candidiasis. The fungicidal nature of anidulafungin coupled with sustained serum concentrations that exceed the MIC90 of virtually all Candida species makes it a very promising systemic antifungal agent.

    ACKNOWLEDGMENTS

    We thank Linda Elliott for excellent assistance in the preparation of the manuscript.

    REFERENCES

    Arevalo, P., A. J. Carrillo-Munoz, J. Salgado, D. Cardenes, S. Brio, G. Quindos, and A. Espinel-Ingroff. 2003. Antifungal activity of the echinocandin anidulafungin (VER002, LY303366) against yeast pathogens: a comparative study with M27-A microdilution method. J. Antimicrob. Chemother. 51:163-166.

    Barry, A. L., M. A. Pfaller, S. D. Brown, A. Espinel-Ingroff, M. A. Ghannoum, C. Knapp, R. P. Rennie, J. H. Rex, and M. G. Rinaldi. 2000. Quality control limits for broth microdilution susceptibility tests of ten antifungal agents. J. Clin. Microbiol. 38:3457-3459.

    Bartizal, K., and F. C. Odds. 2003. Influence of methodological variables on susceptibility testing of caspofungin against Candida species and Aspergillus fumigatus. Antimicrob. Agents Chemother. 47:2100-2107.

    Cuenca-Estrella, M., E. Mellado, T. M. Diaz-Guerra, A. Monzon, and J. L. Rodriguez-Tudela. 2000. Susceptibility of fluconazole-resistant clinical isolates of Candida spp. to echinocandin LY303366, itraconazole and amphotericin B. J. Antimicrob. Chemother. 46:475-477.

    Ernst, E. J., M. E. Klepser, and M. A. Pfaller. 2000. Postantifungal effects of echinocandin, azole, and polyene antifungal agents against Candida albicans and Cryptococcus neoformans. Antimicrob. Agents Chemother. 44:1108-1111.

    Groll, A. H., D. Mickiene, R. Petraitiene, V. Petraitis, C. A. Lyman, J. S. Bacher, S. C. Piscitelli, and T. J. Walsh. 2001. Pharmacokinetic and pharmacodynamic modeling of anidulafungin (LY303366): reappraisal of its efficacy in neutropenic animal models of opportunistic mycoses using optimal plasma sampling. Antimicrob. Agents Chemother. 45:2845-2855.

    Hazen, K. C., and S. A. Howell. 2003. Candida, Cryptococcus, and other yeasts of medical importance, p. 1693-1711. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of clinical microbiology, 8th ed. ASM Press, Washington, D.C.

    Krause, D. S., J. Reinhardt, J. A. Vazquez, A. Reboli, B. P. Goldstein, M. Wible, and T. Henkel. 2004. A phase 2, randomized, dose-ranging study evaluating the safety and efficacy of anidulafungin in invasive candidiasis and candidemia. Antimicrob. Agents Chemother. 48:2021-2024.

    National Committee for Clinical Laboratory Standards. 2002. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard, 2nd ed. M27-A2. National Committee for Clinical Laboratory Standards, Wayne, Pa.

    Odds, F. C., M. Motyl, R. Andrade, J. Bille, E. Canton, M. Cuenca-Estrella, A. Davidson, C. Durussel, D. Ellis, E. Foraker, A. W. Fothergill, M. A. Ghannoum, R. A. Giacobbe, M. Gobernado, R. Handke, M. Laverdiere, W. Lee-Yang, W. G. Merz, L. Ostrosky-Zeicher, J. Pemán, S. Perea, J. R. Perfect, M. A. Pfaller, L. Proia, J. H. Rex, M. G. Rinaldi, J.-L. Rodriguez-Tudela, W. A. Schell, C. Shields, D. A. Sutton, P. E. Verweij, and D. W. Warnock. 2004. Interlaboratory comparison of results of susceptibility testing with caspofungin against Candida and Aspergillus species. J. Clin. Microbiol. 42:3475-3482.

    Ostrosky-Zeichner, L., J. H. Rex, P. G. Pappas, R. J. Hamill, R. A. Larsen, H. W. Horowitz, W. G. Powderly, N. Hyslop, C. A. Kauffman, J. Cleary, J. E. Mangino, and L. Lee. 2003. Antifungal susceptibility survey of 2,000 bloodstream Candida isolates in the United States. Antimicrob. Agents Chemother. 47:3149-3154.

    Petraitiene, R., V. Petraitis, A. H. Groll, M. Candelario, T. Sein, A. Bell, C. A. Lyman, C. L. McMillian, J. Bacher, and T. J. Walsh. 1999. Antifungal activity of LY303366, a novel echinocandin B, in disseminated candidosis in rabbits. Antimicrob. Agents Chemother. 43:2148-2155.

    Pfaller, M. A. 2004. Anidulafungin: an echinocandin antifungal. Expert Opin. Investig. Drugs 13:1183-1197.

    Pfaller, M. A., S. A. Messer, and S. Coffman. 1997. In vitro susceptibilities of clinical yeast isolates to a new echinocandin derivative, LY303366, and other antifungal agents. Antimicrob. Agents Chemother. 41:763-766.

    Pfaller, M. A., S. A. Messer, L. Boyken, C. Rice, S. Tendolkar, R. J. Hollis, and D. J. Diekema. 2003. Caspofungin activity against clinical isolates of fluconazole-resistant Candida. J. Clin. Microbiol. 41:5729-5731.

    Pfaller, M. A., S. A. Messer, L. Boyken, C. Rice, S. Tendolkar, R. J. Hollis, and D. J. Diekema. 2004. Further standardization of broth microdilution methodology for in vitro susceptibility testing of caspofungin against Candida by use of an international collection of more than 3,000 clinical isolates. J. Clin. Microbiol. 42:3117-3119.

    Uzun, O., S. Kocagoz, Y. Cetinkaya, S. Arikan, and S. Unal. 1997. In vitro activity of a new echinocandin, LY-303366, compared with those of amphotericin B and fluconazole against clinical yeast isolates. Antimicrob. Agents Chemother. 41:1156-1157.

    Zhanel, G. G., J. A. Karlowsky, S. A. Zelenitsky, M. A. Turik, and D. J. Hoban. 1998. Susceptibilities of Candida species isolated from the lower gastrointestinal tracts of high-risk patients to the new semisynthetic echinocandin LY303366 and other antifungal agents. Antimicrob. Agents Chemother. 42:2446-2448.

日期:2007年5月10日 - 来自[2005年第43卷第11期]栏目
循环ads

Relationship between MIC and Minimum Sterol 14-Demethylation-Inhibitory Concentration as a Factor in Evaluating Activities of Azoles against Various Fungal Sp

    Division of Oral Infectious Diseases and Immunology, Faculty of Dental Sciences
    Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan
    Department of Bioactive Molecules, National Institute of Infectious Diseases
    Department of Infectious Diseases, Tokyo Women's Medical University, School of Medicine, Tokyo, Japan

    ABSTRACT

    The minimum growth-inhibitory concentrations (MICs) of azole antifungals were compared to their minimum sterol 14-demethylation-inhibitory concentrations (MDICs) for clinical fungal isolates. The ascomycetous Candida yeasts tested were clearly divided into two groups: group I, consisting of C. albicans, C. tropicalis, and C. lusitaniae, had MICs that were much higher than the MDICs, whereas group II, comprising C. glabrata, C. parapsilosis, C. guilliermondii, and C. krusei, had MICs that were approximately equal to the MDICs. In the ascomycetous fungi Aspergillus fumigatus and Sporothrix schenckii, the MICs were indistinguishable from the MDICs. In the basidiomycetous fungi Cryptococcus (Filobasidiella) neoformans, C. curvatus, and Trichosporon asahii, the MICs and the MDICs were practically identical. These results support the notion that there are two distinct classes of fungi differing in their degree of tolerance to sterol 14-demethylation deficiency. These findings have significant implications for both fungal physiology and antifungal chemotherapy.

    INTRODUCTION

    Azole antifungal agents are known to inhibit the activity of cytochrome P45014DM, which catalyzes the 14-demethylation of sterols in ergosterol biosynthesis (21). As a consequence, cells treated with those drugs accumulate 14-methylated sterols in their membranes in place of ergosterol. A notable feature of this change in sterol composition is that it may or may not inhibit cell growth depending on the fungus involved. Thus, our previous work has demonstrated that under conditions of 14-demethylation inhibition, cells of Candida albicans, C. tropicalis, C. guilliermondii, and C. kefyr can proliferate in vitro, while those of C. krusei, C. glabrata, and C. parapsilosis cannot (18). Unfortunately, however, it has remained unclear whether this characteristic varies within each species due to the small number of strains examined.

    In vivo data, however, indicate that azoles are therapeutically effective in C. albicans infection due to the inhibition of 14-demethylation (11). This could be because 14-demethylation-deficient cells are more vulnerable than normal cells to killing by reactive oxygen species (16) or phagocytes (3, 4, 5, 7, 20).

    The most important implication of the above-mentioned findings is that the minimum sterol 14-demethylation-inhibitory concentration (MDIC), rather than the minimum growth-inhibitory concentration (MIC), may be of primary importance for the clinical use of azole drugs. Theoretically at least, if the MIC of a drug is higher than its MDIC for a certain fungus, the drug may suppress infections it causes even at a serum concentration below the MIC (but above the MDIC) as long as host defenses are unimpaired. It is therefore necessary to collect more information about the relationship between MIC and MDIC. In the present study, we provide such data for a number of clinical isolates, including not only Candida species but also other ascomycetous and basidiomycetous fungi.

    MATERIALS AND METHODS

    Fungal strains. Ninety-five clinical isolates of yeast-like fungi, recovered from the bloodstream of patients with fungal infections, consisted of 26 strains of C. albicans, 17 strains of C. tropicalis, 8 strains of C. glabrata, 10 strains of C. parapsilosis, 21 strains of C. guilliermondii, 9 strains of C. krusei (of which 4 are laboratory strains), 2 strains of C. lusitaniae (1), 2 strains of Cryptococcus (Filobasidiella) neoformans, 2 strains of Cryptococcus curvatus (8), and 2 strains of Trichosporon asahii (19). These isolates were identified presumptively by colony morphology on CHROMagar Candida (Becton Dickinson), chlamydospore formation on cornmeal agar, and sugar assimilation patterns with the API ID32C system (BioMerieux SA, Marcy-l'Etoile, France). Species identification was confirmed by direct sequencing of the D1-D2 region of 26S rRNA (6). Five strains of Aspergillus fumigatus isolated from animals were provided by Rui Kano of the School of Veterinary Medicine, Nippon University. Sporothrix schenckii IFM41598, originally isolated from a patient with sporothrichosis, was provided by Kazuko Nishimura of Chiba University.

    MIC and MDIC determination. We have previously devised a simple method capable of measuring the MDIC of an azole drug for those fungi whose viability is unaffected by sterol 14-demethylation deficiency, e.g., C. albicans (17, 18). The method is based on the fact that 14-demethylation deficiency, whether caused by an azole or another mechanism, makes the fungal cells susceptible to growth inhibition by acetate added to the growth medium, probably due to an increase in permeability of the cell membrane (17). Thus, for fungi of this class the MIC of an azole as determined in acetate-supplemented medium (MICAc) is lower than its MIC measured in acetate-free medium and is similar to its MDIC, as estimated by sterol profiling with thin-layer chromatography; hence, the relationship was MIC > MICAc = MDIC (18). In contrast, those fungi which are unable to tolerate 14-demethylation deficiency, e.g., C. krusei, are characterized by the formula MIC = MICAc = MDIC (18). Incidentally, acetate has no effect on sterol demethylation or growth rate at the concentration used. In practice, the determination of MIC and MDIC was carried out as follows. Twofold dilutions of the test drug were made in yeast extract-peptone-glucose (YEPG) medium consisting of 1% yeast extract, 2% polypeptone, and 2% glucose (for MICY, which means MIC measured in YEPG) or YEPG supplemented with 0.24 M sodium acetate (YEPG-Ac) (for MDIC). A 0.1-ml portion of each dilution was mixed in a microplate well with 0.1 ml of a cell suspension in the same medium containing approximately 105 CFU. The plates were incubated at 37°C for 2 days with shaking. The lowest drug concentration that gave a prominent decrease in turbidity was taken to represent the MICY or MDIC of the drug. It should be noted that the MICY obtained in this way is not equivalent to the MIC determined by the routine method described by the National Committee for Clinical Laboratory Standards (9). In the case of S. schenckii, the high susceptibility of this fungus to acetate made the use of the above method for MDIC determination inadequate. Hence, direct estimation of MDIC by sterol profiling with thin-layer chromatography was carried out as previously described (17).

    RESULTS

    Candida species. Our previous study demonstrated that the strains of Candida species examined were divided into two groups with respect to the relationship between MIC and MDIC (18). However, the small number of test strains made it uncertain whether this characteristic was species specific. In the present work, we studied this dichotomy with a much larger number of strains (Table 1). For strains of C. albicans and C. tropicalis, the MICY (>200 μg/ml for fluconazole) was much higher than the MDIC (0.2 to 3.1 μg/ml), as previously reported. In addition, strains of C. lusitaniae were found to belong to this group. On the other hand, the MICY was essentially equal to the MDIC for strains of C. glabrata, C. parapsilosis, and C. krusei, in agreement with the previous results (18). Only C. guilliermondii exhibited an intraspecific heterogeneity: strain IFO0454, used in the previous studies, is a member of the former group, while the 21 strains tested in this work were found to belong to the latter group. These results strongly suggest that intraspecies variation in this MIC-MDIC characteristic is rare. In other words, this characteristic can be regarded as species specific for Candida species.

    Basidiomycetous yeast species. For all strains of the basidiomycetous yeasts C. neoformans, C. curvatus, and Trichosporon asahii tested, the MICY was found to be comparable to the MDIC (Table 1).

    Filamentous fungi. We successfully applied the YEPG-Ac method for MDIC determination to five strains of the ascomycetous mold A. fumigatus, and we found for all of them that the MICY was comparable to the MDIC (Table 1). The ascomycetous fungus S. schenckii is dimorphic and grows either as filaments at 25°C or as yeasts at 37°C (10). Because the growth of this fungus turned out to be severely inhibited by acetate even in the absence of azoles, the use of YEPG-Ac medium for MDIC determination was considered inadequate. We therefore studied growth and sterol composition of cells of this fungus cultured aerobically in YEPG medium at 25°C in the presence of various concentrations of ketoconazole. The azole drug inhibited by more than 80% both cell growth and sterol 14-demethylation at a concentration of 1.25 μg/ml and higher compared to the drug-free control cells (Fig. 1). The same results were obtained with yeast cells grown at 37°C (data not shown). Thus, the MICY and MDIC of ketoconazole were shown to be similar for this organism. In addition, the MICY estimated by optical density was compatible with morphological observations. While cells grew in typically filamentous form without ketoconazole, the presence of the drug at 1.25 μg/ml completely inhibited their growth.

    DISCUSSION

    The results of the present study are consistent with the notion that fungi can be divided into two types based on their tolerance to deficiency in sterol 14-demethylation. Most, if not all, species appear to be homogeneous with respect to this trait, and the species for which the drug MIC and MDIC are comparable to each other appear to be more common than those for which the in vitro MIC is higher than the MDIC. The mechanism underlying the difference in the degree of tolerance to 14-methylated sterols is unknown at present. One possibility is that different degrees of tolerance may be due to the effect of the 14-methyl group on the function of one or more membrane proteins that interact with sterols in membranes.

    C. albicans and C. tropicalis, for which the MICY is higher than the MDIC, are both important opportunistic pathogens, frequently causing systemic mycoses in compromised hosts. Our previous studies have shown that C. albicans cells exhibit increased sensitivity to various antifungal chemicals when their membranes contain 14-methylated sterols in place of ergosterol, probably due to enhanced membrane permeability (13, 15). This observation immediately suggests the possibility that a combination of an azole and an antifungal from another class could act synergistically on fungal cells at least of this type. It is hoped that this may open up a new avenue of antifungal chemotherapy.

    The values of MDICs for Candida species are similar to MICs obtained by the standard NCCLS RPMI method (9, 18) and correlate with the effectiveness of antifungal agents in vivo. We are therefore currently investigating the wider application of the simple MDIC methodology to the azole susceptibility testing of filamentous fungi, such as Aspergillus spp.

    ACKNOWLEDGMENTS

    This work was supported by a grant from the Japan Health Sciences Foundation to O.S. and M.N. and Health Science Research Grants for Research on Emerging and Re-emerging Infectious Diseases, Ministry of Health, Labor and Welfare of Japan to M.N. and K.K.

    Gifts of azole drugs from Pfizer Pharmaceuticals and Janssen Research Foundation are gratefully acknowledged. We thank Kazuko Nishimura of Chiba University and Rui Kano of Nippon University for providing us with fungal strains. We would also like to thank Hiroaki Nakayama, emeritus professor of Kyushu University, and Richard D. Cannon of Otago University for scientific discussion and critical reading of the manuscript.

    REFERENCES

    al-Rawi, N., and K. Kavanagh. 1999. Characterization of yeasts implicated in vulvovaginal candidosis in Irish women. Br. J. Biomed. Sci. 56:99-104.

    Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917.

    De Brabander, M., F. Aerts, J. Van Custem, H. Vanden Bossche, and M. Borgers. 1980. The activity of ketoconazole in mixed cultures of leukocytes and Candida albicans. Sabouraudia 18:197-210.

    Garcha, U. K., E. Brummer, and D. A. Stevens. 1995. Synergy of fluconazole with human monocytes or monocyte-derived macrophages for killing of Candida species. J. Infect. Dis. 172:1620-1623.

    Hazen, K. C., G. Mandell, E. Coleman, and G. Wu. 2000. Influence of fluconazole at subinhibitory concentrations on cell surface hydrophobicity and phagocytosis of Candida albicans. FEMS Microbiol. Lett. 183:89-94.

    Kurtzman, C. P., and C. J. Robnett. 1997. Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA gene. J. Clin. Microbiol. 35:1216-1223.

    Minguez, F., J. E. Lima, M. T. Garcia, and J. Prieto. 1997. Effects of antifungal pretreatment on the susceptibility of Candida albicans to human leukocytes. Chemotherapy 43:346-351.

    Mouligner, D. F., B. Dupont, E. Gueho, M. Baudrimont, L. Imprevisi, F. Provost, and G. Gonzalez-Canali. 1995. Myeloradiculitis due to Cryptococcus curvatus in AIDS. AIDS 9:395-396.

    National Committee for Clinical Laboratory Standards. 2002. Reference method for broth microdilution antifungal susceptibility testing of yeasts. Approved standard M27-A2. National Committee for Clinical Laboratory Standards, Wayne, Pa.

    Rodriguez-Del Valle, N., M. Rosario, and G. Torres-Blasini. 1983. Effects of pH, temperature, aeration and carbon source on the development of the mycelial or yeast forms of Sporothrix schenckii from conidia. Mycopathologia 82:83-88.

    Shigematsu, M. L., J. Uno, and T. Arai. 1981. Correlative studies on in vivo and in vitro effectiveness of ketoconazole (R41400) against Candida albicans infection. Jpn. J. Med. Mycol. 22:195-201.

    Shimokawa, O., Y. Kato, and H. Nakayama. 1986. Accumulation of 14-methyl sterols and defective hyphal growth in Candida albicans. J. Med. Vet. Mycol. 24:327-336.

    Shimokawa, O., Y. Kato, and H. Nakayama. 1986. Increased drug sensitivity in Candida albicans cells accumulating 14-methylated sterols. J. Med. Vet. Mycol. 24:481-483.

    Shimokawa, O., Y. Kato, K. Kawano, and H. Nakayama. 1989. Accumulation of 14-methylergosta-8,24(28)-dien-3,6-diol in 14-demethylation mutants of Candida albicans: genetic evidence for the involvement of 5-desaturase. Biochim. Biophys. Acta 1003:15-19.

    Shimokawa, O., and H. Nakayama. 1989. A Candida albicans mutant conditionally defective in sterol 14-demethylation. J. Med. Vet. Mycol. 27:121-125.

    Shimokawa, O., and H. Nakayama. 1992. Increased sensitivity of Candida albicans cells accumulating 14-methylated sterols to active oxygen: possible relevance to in vivo efficacies of azole antifungal agents. Antimicrob. Agents Chemother. 36:1626-1629.

    Shimokawa, O., and H. Nakayama. 1999. Acetate-mediated growth inhibition in sterol 14-demethylation-deficient cells of Candida albicans. Antimicrob. Agents Chemother. 43:100-105.

    Shimokawa, O., and H. Nakayama. 2000. Estimation of minimum sterol 14-demethylation-inhibitory concentration of azoles in Candida yeasts using acetate-mediated growth inhibition: potential utility in susceptibility testing. J. Clin. Microbiol. 38:2893-2896.

    Tokimatsu, I., R. Karashima, E. Yamagata, Y. Yamakami, H. Nagai, J. Kadota, and M. Nasu. 2003. Pathogenesis of Trichosporon asahii and strategies for infectious control of disseminated trichosporonosis. Jpn. J. Med. Mycol. 44:181-186.

    Tullio, V., A. M. Cuffini, C. De Leo, F. Perrone, and N. A. Carlone. 1996. Interaction of Candida albicans, macrophages and fluconazole: in vitro and ex vivo observations. J. Chemother. 8:438-444.

    Vanden Bossche, H., G. Willemsens, W. Cools, W. F. G. Lauwers, and L. Le Jeune. 1978. Biochemical effects of miconazole on fungi II. Inhibition of ergosterol biosynthesis in Candida albicans. Chem.-Biol. Interact. 21:59-78.

日期:2007年5月10日 - 来自[2005年第43卷第11期]栏目
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