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1.  Revised Definitions of Invasive Fungal Disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group 
Background
Invasive fungal diseases are important causes of morbidity and mortality. Clarity and uniformity in defining these infections are important factors in improving the quality of clinical studies. A standard set of definitions strengthens the consistency and reproducibility of such studies.
Methods
After the introduction of the original European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group definitions, advances in diagnostic technology and the recognition of areas in need of improvement led to a revision of this document. The revision process started with a meeting of participants in 2003, to decide on the process and to draft the proposal. This was followed by several rounds of consultation until a final draft was approved in 2005. This was made available for 6 months to allow public comment, and then the manuscript was prepared and approved.
Results
The revised definitions retain the original classifications of “proven,” “probable,” and “possible” invasive fungal disease, but the definition of “probable” has been expanded, whereas the scope of the category “possible” has been diminished. The category of proven invasive fungal disease can apply to any patient, regardless of whether the patient is immunocompromised, whereas the probable and possible categories are proposed for immunocompromised patients only.
Conclusions
These revised definitions of invasive fungal disease are intended to advance clinical and epidemiological research and may serve as a useful model for defining other infections in high-risk patients.
doi:10.1086/588660
PMCID: PMC2671227  PMID: 18462102
2.  Treatment options in Invasive Aspergillosis 
doi:10.1007/s40506-014-0016-2
PMCID: PMC4200583  PMID: 25328449
invasive aspergillosis; antifungals; immunotherapy; neutropenia
3.  A Role for LHC1 in Higher Order Structure and Complement Binding of the Cryptococcus neoformans Capsule 
PLoS Pathogens  2014;10(5):e1004037.
Polysaccharide capsules are important virulence factors for many microbial pathogens including the opportunistic fungus Cryptococcus neoformans. In the present study, we demonstrate an unusual role for a secreted lactonohydrolase of C. neoformans, LHC1 in capsular higher order structure. Analysis of extracted capsular polysaccharide from wild-type and lhc1Δ strains by dynamic and static light scattering suggested a role for the LHC1 locus in altering the capsular polysaccharide, both reducing dimensions and altering its branching, density and solvation. These changes in the capsular structure resulted in LHC1-dependent alterations of antibody binding patterns, reductions in human and mouse complement binding and phagocytosis by the macrophage-like cell line J774, as well as increased virulence in mice. These findings identify a unique molecular mechanism for tertiary structural changes in a microbial capsule, facilitating immune evasion and virulence of a fungal pathogen.
Author Summary
Polysaccharide capsules are important virulence factors in pathogenic microbes that provide a protective coat against host immunity. Cryptococcus neoformans is a pathogenic encapsulated yeast that is a major opportunistic infection, causing approximately 600,000 cases of meningitis per year in AIDS patients globally, and whose polysaccharide capsule is a major virulence factor. While extensive work has detailed the chemical components forming the cryptococcal capsule, the molecular events leading to the higher order assembly of the capsule, and its consequences on immune subterfuge remain unknown. In the present studies we used a proteomics method to identify a novel hydrolytic enzyme, lactonohydrolase (Lhc1) and used a variety of biophysical methods including dynamic and static light scattering as well as motility studies to show that extracted capsular polysaccharide undergoes remodeling in a LHC1-dependent fashion. This results in a more tightly compacted capsular structure that alters binding of anti-capsular antibodies and reduces binding by both human as well as mouse serum complement. Furthermore, LHC1-dependent capsular alterations serve to increase the virulence of the fungus in a mouse model, suggesting a novel role for this class of enzyme in capsular remodeling and immune evasion in microbial pathogenesis.
doi:10.1371/journal.ppat.1004037
PMCID: PMC4006888  PMID: 24789368
4.  Anti-granulocyte-macrophage colony stimulating factor autoantibodies in patients with cryptococcal meningitis 
Background
Cryptococcal meningitis has been described in immunocompromised patients as well as in those for whom no immune defect has been identified. GM-CSF regulates the function of phagocytes and pulmonary alveolar macrophages, critical elements in cryptococcal control.
Methods
We performed clinical histories, immunological evaluation, and anticytokine autoantibody screening in 4 current patients with cryptococcal meningitis, and identified and tested 103 archived plasma/CSF samples from patients with cryptococcal meningitis. We assessed the ability of anti-GM-CSF autoantibody containing plasmas to inhibit GM-CSF signaling.
Results
We recognized anti-GM-CSF autoantibodies in an otherwise healthy female with cryptococcal meningitis who later developed pulmonary alveolar proteinosis. Her diagnosis prompted screening of patients with cryptococcal meningitis for anticytokine autoantibodies. We identified 7 HIV uninfected patients with cryptococcal meningitis who tested positive for high-titer anti-GM-CSF autoantibodies. Two of the 7 later developed evidence of PAP. Plasma from all patients prevented GM-CSF-induced STAT-5 phosphorylation and MIP-1α production in normal PBMC. This effect was limited to their IgG fraction.
Conclusions
Anti-GM-CSF autoantibodies are associated with some cases of cryptococcal meningitis in otherwise immunocompetent patients. These cases need not have associated pulmonary alveolar proteinosis.
doi:10.4049/jimmunol.1202526
PMCID: PMC3675663  PMID: 23509356
5.  Transcriptional Profiling of Candida glabrata during Phagocytosis by Neutrophils and in the Infected Mouse Spleen 
Infection and Immunity  2013;81(4):1325-1333.
Expression microarray analysis of Candida glabrata following phagocytosis by human neutrophils was performed, and results were compared with those from C. glabrata incubated under conditions of carbohydrate or nitrogen deprivation. Twenty genes were selected to represent the major cell processes altered by phagocytosis or nutrient deprivation. Quantitative real-time PCR (qRT-PCR) with TaqMan chemistry was used to assess expression of the same genes in spleens of mice infected intravenously with Candida glabrata. The results in spleen closely paralleled gene expression in neutrophils or following carbohydrate deprivation. Fungal cells responded by upregulating alternative energy sources through gluconeogenesis, glyoxylate cycle, and long-chain fatty acid metabolism. Autophagy was likely employed to conserve intracellular resources. Aspartyl protease upregulation occurred and may represent defense against attacks on cell wall integrity. Downregulated genes were in the pathways of protein and ergosterol synthesis. Upregulation of the sterol transport gene AUS1 suggested that murine cholesterol may have been used to replace ergosterol, as has been reported in vitro. C. glabrata isolates in spleens of gp91phox−/− knockout mice with reduced oxidative phagocyte defenses were grossly similar although with a reduced level of response. These results are consistent with reported results of other fungi responding to phagocytosis, indicating that a rapid shift in metabolism is required for growth in a carbohydrate-limited intracellular environment.
doi:10.1128/IAI.00851-12
PMCID: PMC3639592  PMID: 23403555
6.  Microevolution During Serial Mouse Passage Demonstrates FRE3 as a Virulence Adaptation Gene in Cryptococcus neoformans 
mBio  2014;5(2):e00941-14.
ABSTRACT
Passage in mice of opportunistic pathogens such as Cryptococcus neoformans is known to increase virulence, but little is known about the molecular mechanisms involved in virulence adaptation. Serial mouse passage of nine environmental strains of serotype A C. neoformans identified two highly adapted virulent strains that showed a 4-fold reduction in time to death after four passages. Transcriptome sequencing expression studies demonstrated increased expression of a FRE3-encoded iron reductase in the two strains but not in a control strain that did not demonstrate increased virulence during mouse passage. FRE3 was shown to express an iron reductase activity and to play a role in iron-dependent growth of C. neoformans. Overexpression of FRE3 in the two original environmental strains increased growth in the macrophage cell line J774.16 and increased virulence. These data demonstrate a role for FRE3 in the virulence of C. neoformans and demonstrate how the increased expression of such a “virulence acquisition gene” during the environment-to-mammal transition, can optimize the virulence of environmental strains in mammalian hosts.
IMPORTANCE
Cryptococcus neoformans is a significant global fungal pathogen that also resides in the environment. Recent studies have suggested that the organism may undergo microevolution in the host. However, little is known about the permitted genetic changes facilitating the adaptation of environmental strains to mammalian hosts. The present studies subjected environmental strains isolated from several metropolitan areas of the United States to serial passages in mice. Transcriptome sequencing expression studies identified the increased expression of an iron reductase gene, FRE3, in two strains that adapted in mice to become highly virulent, and overexpression of FRE3 recapitulated the increased virulence after mouse passage. Iron reductase in yeast is important to iron uptake in a large number of microbial pathogens. These studies demonstrate the capacity of C. neoformans to show reproducible changes in the expression levels of small numbers of genes termed “virulence adaptation genes” to effectively increase pathogenicity during the environment-to-mammal transition.
doi:10.1128/mBio.00941-14
PMCID: PMC3977352  PMID: 24692633
7.  Anti-Granulocyte-Macrophage Colony-Stimulating Factor Autoantibodies Are a Risk Factor for Central Nervous System Infection by Cryptococcus gattii in Otherwise Immunocompetent Patients 
mBio  2014;5(2):e00912-14.
ABSTRACT
Cryptococcosis is caused by either Cryptococcus neoformans or C. gattii. While cryptococcal meningoencephalitis is caused mostly by C. neoformans in immunocompromised patients, the risk factors remain unclear for patients with no known immune defect. Recently, anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibodies were detected in the plasma of seven “immunocompetent” cryptococcosis patients, and the cryptococcal strains from these patients were reported as C. neoformans (three strains), C. gattii (one strain), and Cryptococcus (three strains not identified to the species level). We identified all three strains that had not been identified to the species level as C. gattii. Notably, the three strains that were reported as C. neoformans but were unavailable for species confirmation originated from Sothern California and Thailand where C. gattii is endemic. Most clinical laboratories designate C. neoformans without distinguishing between the two species; hence, these three strains could have been C. gattii. Since C. gattii infects more immunocompetent patients than C. neoformans, we pursued the possibility that this antibody may be more prevalent in patients infected with C. gattii than in those infected with C. neoformans. We screened the plasma of 20 healthy controls and 30 “immunocompetent” patients with cryptococcal meningoencephalitis from China and Australia (multiple ethnicities). Anti-GM-CSF autoantibodies were detected only in the plasma of seven patients infected by C. gattii and one healthy volunteer and in none infected by C. neoformans. While plasma from these C. gattii patients completely prevented GM-CSF-induced p-STAT5 in normal human peripheral blood mononuclear cells (PBMCs), plasma from one healthy volunteer positive for anti-GM-CSF autoantibodies caused only partial blockage. Our results suggest that anti-GM-CSF autoantibodies may predispose otherwise immunocompetent individuals to meningoencephalitis caused by C. gattii but not necessarily to that caused by C. neoformans.
IMPORTANCE
Cryptococcal meningoencephalitis is the most serious central nervous system (CNS) infection caused by Cryptococcus neoformans or C. gattii. Cryptococcus primarily infects immunocopromised patients but is also sporadically encountered in otherwise “immunocompetent” patients with no known risk. In a recent study, anti-GM-CSF autoantibodies were detected in the plasma of seven otherwise immunocompetent patients with cryptococcal meningoencephalitis. Four of seven (57%) cryptococcal isolates from these patients were identified as C. gattii, while three strains were unavailable for species confirmation. We collected plasma from 30 otherwise healthy patients with CNS cryptococcosis in China and Australia (multiethnic) and analyzed the samples for the presence of anti-GM-CSF autoantibodies. The results suggest that anti-GM-CSF autoantibodies are a risk factor for CNS infection by C. gattii but not C. neoformans. GM-CSF may have a specific role in host defense against C. gattii, thereby elevating the importance of determining the level of anti-GM-CSF autoantibodies which can impact clinical management.
doi:10.1128/mBio.00912-14
PMCID: PMC3967522  PMID: 24643864
8.  STB5 Is a Negative Regulator of Azole Resistance in Candida glabrata 
The opportunistic yeast pathogen Candida glabrata is recognized for its ability to acquire resistance during prolonged treatment with azole antifungals (J. E. Bennett, K. Izumikawa, and K. A. Marr. Antimicrob. Agents Chemother. 48:1773–1777, 2004). Resistance to azoles is largely mediated by the transcription factor PDR1, resulting in the upregulation of ATP-binding cassette (ABC) transporter proteins and drug efflux. Studies in the related yeast Saccharomyces cerevisiae have shown that Pdr1p forms a heterodimer with another transcription factor, Stb5p. In C. glabrata, the open reading frame (ORF) designated CAGL0I02552g has 38.8% amino acid identity with STB5 (YHR178w) and shares an N-terminal Zn2Cys6 binuclear cluster domain and a fungus-specific transcriptional factor domain, prompting us to test for homologous function and a possible role in azole resistance. Complementation of a Δyhr178w (Δstb5) mutant with CAGL0I02552g resolved the increased sensitivity to cold, hydrogen peroxide, and caffeine of the mutant, for which reason we designated CAGl0I02552g CgSTB5. Overexpression of CgSTB5 in C. glabrata repressed azole resistance, whereas deletion of CgSTB5 caused a modest increase in resistance. Expression analysis found that CgSTB5 shares many transcriptional targets with CgPDR1 but, unlike the latter, is a negative regulator of pleiotropic drug resistance, including the ABC transporter genes CDR1, PDH1, and YOR1.
doi:10.1128/AAC.01278-12
PMCID: PMC3553707  PMID: 23229483
9.  The Use of HAART Is Associated With Decreased Risk of Death During Initial Treatment of Cryptococcal Meningitis in Adults in Botswana 
Objective
The objective of this study was to evaluate outcomes among adults with a first episode of cryptococcal meningitis (CM), comparing those on highly active antiretroviral therapy (HAART) with those not on HAART.
Methods
We conducted a prospective cohort study among HIV-infected adults (aged 18 years and older) with a first episode of CM at the Princess Marina Hospital, in Gaborone, Botswana. The proportions surviving to discharge were compared. Logistic regression was used to evaluate the relationship between HAART use and risk of death in the hospital, adjusting for potential confounders.
Results
Ninety-two patients [median CD4 41 cells/mm3 (inter-quartile range 22–85)] were included, 26 of whom were on HAART at the time that they developed CM. The in-hospital mortality was lower among those on HAART {2 of 26 (8%) vs 14 of 66 (21%); odds ratio = 0.36 [95% confidence interval (CI) 0.09 to 1.49]}, and this result was statistically significant after adjustment for male sex and tuberculosis [adjusted odds ratio = 0.19 (95% CI 0.04 to 1.00)].
Conclusions
HAART use at the time of a first admission with CM is associated with decreased risk of death during the acute phase of disease. Reasons for this association should be explored.
doi:10.1097/QAI.0b013e318183181e
PMCID: PMC3704195  PMID: 18769344
cryptococcal meningitis; HAART; Africa; cohort study
10.  Disseminated Microsporidiosis in an Immunosuppressed Patient 
Emerging Infectious Diseases  2012;18(7):1155-1158.
We report a case of disseminated microsporidiosis in a patient with multiple myeloma who had received an allogeneic stem cell transplant requiring substantial immunosuppression. The causative organism was identified as Tubulinosema acridophagus, confirming this genus of microsporidia as a novel human pathogen.
doi:10.3201/eid1807.120047
PMCID: PMC3376806  PMID: 22709509
allogeneic stem cell transplantation; microsporidia; multiple myeloma; disseminated microsporidiosis; fungi; immunosuppressed; Tubulinosema acridophagus; novel human pathogen
11.  Evaluation of reference genes for real-time quantitative PCR studies in Candida glabrata following azole treatment 
BMC Molecular Biology  2012;13:22.
Background
The selection of stable and suitable reference genes for real-time quantitative PCR (RT-qPCR) is a crucial prerequisite for reliable gene expression analysis under different experimental conditions. The present study aimed to identify reference genes as internal controls for gene expression studies by RT-qPCR in azole-stimulated Candida glabrata.
Results
The expression stability of 16 reference genes under fluconazole stress was evaluated using fold change and standard deviation computations with the hkgFinder tool. Our data revealed that the mRNA expression levels of three ribosomal RNAs (RDN5.8, RDN18, and RDN25) remained stable in response to fluconazole, while PGK1, UBC7, and UBC13 mRNAs showed only approximately 2.9-, 3.0-, and 2.5-fold induction by azole, respectively. By contrast, mRNA levels of the other 10 reference genes (ACT1, EF1α, GAPDH, PPIA, RPL2A, RPL10, RPL13A, SDHA, TUB1, and UBC4) were dramatically increased in C. glabrata following antifungal treatment, exhibiting changes ranging from 4.5- to 32.7-fold. We also assessed the expression stability of these reference genes using the 2-ΔΔCT method and three other software packages. The stability rankings of the reference genes by geNorm and the 2-ΔΔCT method were identical to those by hkgFinder, whereas the stability rankings by BestKeeper and NormFinder were notably different. We then validated the suitability of six candidate reference genes (ACT1, PGK1, RDN5.8, RDN18, UBC7, and UBC13) as internal controls for ten target genes in this system using the comparative CT method. Our validation experiments passed for all six reference genes analyzed except RDN18, where the amplification efficiency of RDN18 was different from that of the ten target genes. Finally, we demonstrated that the relative quantification of target gene expression varied according to the endogenous control used, highlighting the importance of the choice of internal controls in such experiments.
Conclusions
We recommend the use of RDN5.8, UBC13, and PGK1 alone or the combination of RDN5.8 plus UBC13 or PGK1 as reference genes for RT-qPCR analysis of gene expression in C. glabrata following azole treatment. In contrast, we show that ACT1 and other commonly used reference genes (GAPDH, PPIA, RPL13A, TUB1, etc.) were not validated as good internal controls in the current model.
doi:10.1186/1471-2199-13-22
PMCID: PMC3482582  PMID: 22747760
Candida glabrata; Azole resistance gene; Fluconazole; hkgFinder; Housekeeping gene; Reference gene; RT-qPCR
12.  Hallucinations during Voriconazole Therapy 
As part of a prospective natural history cohort study of voriconazole toxicity, we describe the characteristics of 12 of 72 voriconazole-treated patients who experienced hallucinations from March 2006 through November 2007. Hallucinations associated with voriconazole use are not uncommon. Doctors should be aware of this complication, and the recipients of the drug should be reassured that the hallucinations are an effect of the drug.
doi:10.1086/588844
PMCID: PMC2727751  PMID: 18491963
13.  Hematogenously Disseminated Skin Disease Caused by Mucor velutinosus in a Patient with Acute Myeloid Leukemia▿ 
Journal of Clinical Microbiology  2011;49(7):2728-2732.
We report here a case of disseminated skin infection caused by Mucor velutinosus, a recently described new species. We believe this to be the first published report of a clinical case of mucormycosis due to M. velutinosus, as well as a rare case of dissemination from a deep site to skin.
doi:10.1128/JCM.00387-11
PMCID: PMC3147817  PMID: 21543575
14.  Microarray and Molecular Analyses of the Azole Resistance Mechanism in Candida glabrata Oropharyngeal Isolates▿  
DNA microarrays were used to analyze Candida glabrata oropharyngeal isolates from seven hematopoietic stem cell transplant recipients whose isolates developed azole resistance while the recipients received fluconazole prophylaxis. Transcriptional profiling of the paired isolates revealed 19 genes upregulated in the majority of resistant isolates compared to their paired susceptible isolates. All seven resistant isolates had greater than 2-fold upregulation of C. glabrata PDR1 (CgPDR1), a master transcriptional regulator of the pleiotropic drug resistance (PDR) network, and all seven resistant isolates showed upregulation of known CgPDR1 target genes. The altered transcriptome can be explained in part by the observation that all seven resistant isolates had acquired a single nonsynonymous mutation in their CgPDR1 open reading frame. Four mutations occurred in the regulatory domain (L280P, L344S, G348A, and S391L) and one in the activation domain (G943S), while two mutations (N764I and R772I) occurred in an undefined region. Association of azole resistance and the CgPDR1 mutations was investigated in the same genetic background by introducing the CgPDR1 sequences from one sensitive isolate and five resistant isolates into a laboratory azole-hypersusceptible strain (Cgpdr1 strain) via integrative transformation. The Cgpdr1 strain was restored to wild-type fluconazole susceptibility when transformed with CgPDR1 from the susceptible isolate but became resistant when transformed with CgPDR1 from the resistant isolates. However, despite the identical genetic backgrounds, upregulation of CgPDR1 and CgPDR1 target genes varied between the five transformants, independent of the domain locations in which the mutations occurred. In summary, gain-of-function mutations in CgPDR1 contributed to the clinical azole resistance, but different mutations had various degrees of impact on the CgPDR1 target genes.
doi:10.1128/AAC.00535-10
PMCID: PMC2916311  PMID: 20547810
15.  A cluster of nosocomial Legionnaire’s disease linked to a contaminated hospital decorative water fountain 
Background
Nosocomial outbreaks of Legionnaire’s disease have been linked to contaminated water in hospitals. Immunocompromised patients are particularly vulnerable and, when infected, have a high mortality rate. We report the investigation of a cluster of nosocomial pneumonia due to Legionella pneumophila serogroup 1 that occurred among patients on our stem cell transplantation unit.
Methods
We conducted a record review to identify common points of potential exposure, followed by environmental and water sampling for Legionella spp. from those sources. We used an air sampler in an attempt to detect aerosolized Legionella, and pulsed-field gel electrophoresis to compare clinical and environmental isolates.
Results
The most likely sources identified were the water supply in the patients’ rooms and a decorative fountain in the radiation oncology suite. Samples from the patients’ rooms did not grow Legionella species. Cultures of the fountain, which had been restarted 4 months earlier after being shut off for 5 months, yielded L. pneumophila serogroup 1. The isolates from both patients and the fountain were identical by pulsed-field gel electrophoresis. Both patients developed pneumonia within 10 days of completing radiation therapy, and each reported having observed the fountain at close range. Both patients’ infections were identified early and treated promptly, and both recovered.
Conclusions
This cluster was caused by contamination of a decorative fountain despite its being equipped with a filter and ozone generator. Fountains are a potential source of nosocomial Legionnaire’s disease despite standard maintenance and sanitizing measures. In our opinion, fountains present unacceptable risk in hospitals serving immunocompromised patients.
doi:10.1086/598855
PMCID: PMC2886954  PMID: 19580436
Legionnaire’s; Legionella; nosocomial; fountain; water feature
16.  Management of mycoses in neutropenic patients: a brief history, 1960–2008 
Journal of Antimicrobial Chemotherapy  2009;63(Suppl 1):i23-i26.
The career that this symposium honours, that of Ben E. de Pauw, MD, PhD, could be said to have begun in 1970 upon his graduation from the University of Amsterdam. However, it was his move to Nijmegen in 1975 to finish his PhD studies that really began the career in which he forged expertise in haematology, oncology, immunology and infectious diseases into one spectacular career that resulted in more than 200 publications.
doi:10.1093/jac/dkp079
PMCID: PMC3143421  PMID: 19372177
fungi; cancer; infections
17.  Defining Responses to Therapy and Study Outcomes in Clinical Trials of Invasive Fungal Diseases: Mycoses Study Group and European Organization for Research and Treatment of Cancer Consensus Criteria 
Invasive fungal diseases (IFDs) have become major causes of morbidity and mortality among highly immunocompromised patients. Authoritative consensus criteria to diagnose IFD have been useful in establishing eligibility criteria for antifungal trials. There is an important need for generation of consensus definitions of outcomes of IFD that will form a standard for evaluating treatment success and failure in clinical trials. Therefore, an expert international panel consisting of the Mycoses Study Group and the European Organization for Research and Treatment of Cancer was convened to propose guidelines for assessing treatment responses in clinical trials of IFDs and for defining study outcomes. Major fungal diseases that are discussed include invasive disease due to Candida species, Aspergillus species and other molds, Cryptococcus neoformans, Histoplasma capsulatum, and Coccidioides immitis. We also discuss potential pitfalls in assessing outcome, such as conflicting clinical, radiological, and/or mycological data and gaps in knowledge.
doi:10.1086/590566
PMCID: PMC2671230  PMID: 18637757
18.  Cryptococcosis 
Western Journal of Medicine  1980;132(2):150.
PMCID: PMC1271997  PMID: 18748555
19.  Stability of Amphotericin B in Infusion Bottles 
Intravenous solutions of amphotericin B in 5% dextrose water with or without hydrocortisone or heparin demonstrated no appreciable loss of activity when exposed to fluorescent light for up to 24 h at 25 C (room temperature).
PMCID: PMC444613  PMID: 4793885
20.  Fluconazole Treatment Is Effective against a Candida albicans erg3/erg3 Mutant In Vivo Despite In Vitro Resistance 
Candida albicans ERG3 encodes a sterol C5,6-desaturase which is essential for synthesis of ergosterol. Defective sterol C5,6 desaturation has been considered to be one of the azole resistance mechanisms in this species. However, the clinical relevance of this resistance mechanism is still unclear. In this study, we created a C. albicans erg3/erg3 mutant by the “Ura-blaster” method and confirmed the expected azole resistance using standard in vitro testing and the presence of ergosta-7,22-dien-3β-ol instead of ergosterol. For in vivo studies, a wild-type URA3 was placed back into its native locus in the erg3 homozygote to avoid positional effects on URA3 expression. Defective hyphal formation of the erg3 homozygote was observed not only in vitro but in kidney tissues. A marked attenuation of virulence was shown by the longer survival and the lower kidney burdens of mice inoculated with the reconstituted Ura+ erg3 homozygote relative to the control. To assess fluconazole efficacy in a murine model of disseminated candidiasis, inoculum sizes of the control and the erg3 homozygote were chosen which provided a similar organ burden. Under these conditions, fluconazole was highly effective in reducing the organ burden in both groups. This study demonstrates that an ERG3 mutation causing inactivation of sterol C5,6-desaturase cannot confer fluconazole resistance in vivo by itself regardless of resistance measured by standard in vitro testing. The finding questions the clinical significance of this resistance mechanism.
doi:10.1128/AAC.50.2.580-586.2006
PMCID: PMC1366932  PMID: 16436713
21.  Candida glabrata PDR1, a Transcriptional Regulator of a Pleiotropic Drug Resistance Network, Mediates Azole Resistance in Clinical Isolates and Petite Mutants 
Candida glabrata, a yeast with intrinsically low susceptibility to azoles, frequently develops increased azole resistance during prolonged treatment. Transposon mutagenesis revealed that disruption of CgPDR1 resulted in an 8- to 16-fold increase in fluconazole susceptibility of C. glabrata. CgPDR1 is a homolog of Saccharomyces cerevisiae PDR1, which encodes a transcriptional regulator of multidrug transporters. Northern blot analyses indicated that CgPDR1 regulated both constitutive and drug-induced expression of CgCDR1, a multidrug transporter gene. In agreement with the Northern analysis, the Cgpdr1 mutant had increased rhodamine accumulation, in contrast to the decreased accumulation in the CgPDR1-overexpressing strain. Northern analyses also indicated the importance of CgPDR1 in fluconazole resistance arising during therapy. Two clinically resistant isolates had higher expression of CgPDR1 and CgCDR1 compared to their paired susceptible isolates. Integrative transformation of CgPDR1 from the two resistant isolates converted the Cgpdr1 mutant into azole-resistant strains with upregulated CgPDR1 expression. Two different amino acid substitutions, W297S in one isolate and F575L in the other, accounted for the upregulated CgPDR1 expression and the resistance. Finally, CgPDR1 was shown to be required for the azole resistance due to mitochondrial deficiency. Thus, CgPDR1 encodes a transcriptional regulator of a pleiotropic drug resistance network and contributes to the azole resistance of clinical isolates and petite mutants.
doi:10.1128/AAC.50.4.1384-1392.2006
PMCID: PMC1426987  PMID: 16569856
22.  Candida glabrata erg1 Mutant with Increased Sensitivity to Azoles and to Low Oxygen Tension 
A Candida glabrata erg1 (Cgerg1) mutant, CgTn201S, was identified by transposon mutagenesis and by increased fluconazole susceptibility. CgERG1 encodes a 489-amino-acid protein which, on the basis of its homology with Saccharomyces cerevisiae ERG1, is a squalene epoxidase essential for ergosterol synthesis. Interruption following codon 475 of CgErg1p decreased the ergosterol content by 50%; caused accumulation of the squalene precursor; increased the levels of susceptibility to fluconazole, itraconazole, and terbinafine; increased the level of resistance to amphotericin B; increased the levels of rhodamine 6G and [3H]-fluconazole uptake; reduced the level of growth; and blocked growth under conditions of low oxygen tension. In addition, CgTn201S efficiently took up exogenous cholesterol from cholesterol-containing serum. Cholesterol constituted 34% of the extractable sterols in CgTn201S when it was grown aerobically on serum-containing medium. Under the same conditions, C. albicans contained only 0.1 to 1.2% cholesterol. Exogenous sterols also restored growth under conditions of low oxygen tension. Finally, complementation of the Cgerg1 mutation restored the levels of [3H]fluconazole uptake and drug susceptibility to wild-type levels.
doi:10.1128/AAC.48.7.2483-2489.2004
PMCID: PMC434157  PMID: 15215098
23.  Mechanism of Increased Fluconazole Resistance in Candida glabrata during Prophylaxis 
Candida glabrata can become resistant to fluconazole, causing persistent colonization and invasive infection during prolonged exposure to the drug. To determine the mechanism of resistance in this setting, weekly oropharyngeal cultures for C. glabrata were obtained over a 2-year period from hematopoietic stem cell transplant recipients who were receiving fluconazole prophylaxis. In 20 patients from whom at least two isolates of the same karyotype were obtained more than two weeks apart, fluconazole MICs doubled every 31 days on average. The mechanism of fluconazole resistance in isolates from the 14 of the 20 patients studied in whom MICs changed at least fourfold was studied. Cellular resistance was accompanied by increased drug efflux as measured by decreased accumulation of fluconazole and rhodamine 6G and increased abundance of transcripts from two drug transporters, CgCDR1 and PDH1. The rapidity and regularity of the rising resistance indicated that C. glabrata is able to upregulate drug efflux without losing the ability to maintain colonization.
doi:10.1128/AAC.48.5.1773-1777.2004
PMCID: PMC400565  PMID: 15105134
24.  Efficacies of Fluconazole, Caspofungin, and Amphotericin B in Candida glabrata-Infected p47phox−/− Knockout Mice 
Candida glabrata is the second leading cause of adult candidemia, resulting in high mortality. Amphotericin B is considered the treatment of choice, while the efficacy of fluconazole is controversial and caspofungin efficacy is unknown. To ascertain drug efficacy in vivo, the utility of a murine model of C. glabrata infection was investigated. C. glabrata was found to cause progressive, lethal infection when injected intravenously into C57BL/6 mice with reduced oxidative microbicidal capacity due to knockout of the p47phox gene. Spleen and kidney organ CFU counts were determined in groups of mice 2 days after the mice completed 6 days of daily intraperitoneal drug treatment, which began on the day of infection. Daily injections of fluconazole at 80 mg/kg did not reduce spleen or kidney CFU counts after infection with C. glabrata strains having in vitro fluconazole MICs of 2, 32, or 256 μg/ml compared to saline-treated controls. However, this fluconazole regimen reduced spleen CFU counts in mice infected with Candida albicans, an infection that is known to be responsive to fluconazole. Caspofungin at 5 mg/kg and amphotericin B at 5 mg/kg were both effective in reducing fungal burden in spleens and kidneys of C. glabrata-infected mice. Ten mice treated for 6 days with caspofungin at 1 mg/kg survived for 15 days, though all 10 saline-injected mice died or were so ill that they had to be sacrificed by 96 h postinfection. This murine model provided evidence of the efficacy of amphotericin B and caspofungin but not of fluconazole against C. glabrata infection.
doi:10.1128/AAC.46.5.1240-1245.2002
PMCID: PMC127175  PMID: 11959551
25.  Genetic Analysis of Azole Resistance in the Darlington Strain of Candida albicans 
Antimicrobial Agents and Chemotherapy  2000;44(11):2985-2990.
High-level azole resistance in the Darlington strain of Candida albicans was investigated by gene replacement in C. albicans and expression in Saccharomyces cerevisiae. We sequenced the ERG11 gene, which encodes the sterol C14α-demethylase, from our copy of the Darlington strain. Both alleles contained the histidine for tyrosine substitution at position 132 (Y132H) reported in Darlington by others, but we also found a threonine-for-isoleucine substitution (I471T) not previously reported in the C. albicans ERG11. The encoded I471T change in amino acids conferred azole resistance when overexpressed alone and increased azole resistance when added to the Y132H amino acid sequence in an S. cerevisiae expression system. Replacement of one copy of ERG11 in an azole-susceptible strain of C. albicans with a single copy of the Darlington ERG11 resulted in expression of the integrated copy and a modest increase in azole resistance. The profound azole resistance of the Darlington strain is the result of multiple mutations.
PMCID: PMC101590  PMID: 11036010

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