In JoVE (1)

Other Publications (180)

Articles by Cornelia Lass-Flörl in JoVE

Other articles by Cornelia Lass-Flörl on PubMed

Mechanism of Human Immunodeficiency Virus-induced Complement Expression in Astrocytes and Neurons

Journal of Virology. Apr, 2002  |  Pubmed ID: 11884542

The cerebral complement system is hypothesized to contribute to neurodegeneration in the pathogenesis of AIDS-associated neurological disorders. Our former results have shown that the human immunodeficiency virus (HIV) strongly induces the synthesis of complement factor C3 in astrocytes. This upregulation explains in vivo data showing elevated complement levels in the cerebrospinal fluid of patients with AIDS-associated neurological symptoms. Since inhibition of complement synthesis and activation in the brain may represent a putative therapeutic goal to prevent virus-induced damage, we analyzed in detail the mechanisms of HIV-induced modulation of C3 expression. HIV-1 increased the C3 levels in astrocyte culture supernatants from 30 to up to 400 ng/ml; signal transduction studies revealed that adenylate cyclase activation with upregulation of cyclic AMP is the central signaling pathway to mediate that increase. Furthermore, activity of protein kinase C is necessary for HIV induction of C3, since inhibition of protein kinase C by prolonged exposure to the phorbol ester tetradecanoyl phorbol acetate partly abolished the HIV effect. The cytokines tumor necrosis factor alpha and gamma interferon were not involved in mediating the HIV-induced C3 upregulation, since neutralizing antibodies had no effect. Besides whole HIV virions, the purified viral proteins Nef and gp41 are biologically active in upregulating C3, whereas Tat, gp120, and gp160 were not able to modulate C3 synthesis. Further experiments revealed that neurons were also able to respond on incubation with HIV with increased C3 synthesis, although the precise pattern was slightly different from that in astrocytes. This strengthens the hypothesis that HIV-induced complement synthesis represents an important mechanism for the pathogenesis of AIDS in the brain.

Routine Handling of Propofol Prevents Contamination As Effectively As Does Strict Adherence to the Manufacturer's Recommendations

Canadian Journal of Anaesthesia = Journal Canadien D'anesthesie. Apr, 2002  |  Pubmed ID: 11927472

Propofol is a potential vector of infection, because it contains no preservative. Thus, the manufacturer's specific recommendations for preparing injections or infusions go beyond the guidelines commonly used in our operating rooms for preparing other iv drugs. The purpose of the present study was to determine whether in the daily routine of an operating theatre a modified propofol handling technique can prevent contamination as effectively as do the manufacturer's handling recommendations.

Antifungal Properties of 5-hydroxytryptamine (serotonin) Against Aspergillus Spp. in Vitro

International Journal of Medical Microbiology : IJMM. Mar, 2002  |  Pubmed ID: 12008920

This study shows that 5-hydroxytrypatmine (5-HT, serotonin) is fungicidal towards conidia and hyphae of clinical isolates of Aspergillus (A.) fumigatus, A. flavus and A. terreus. The minimal fungicidal concentrations for Aspergillus conidia and hyphae ranged between 14.68 to 117.5 mM and 29.37 to 235 mM during 24 and 48 h of incubation. Several serotonin receptor antagonists (5-HT2, 5-HT3) studied in vitro did not influence antifungal activity.

In Vitro Studies on the Activity of Amphotericin B and Lipid-based Amphotericin B Formulations Against Aspergillus Conidia and Hyphae

Mycoses. Jun, 2002  |  Pubmed ID: 12100533

The minimum inhibitory concentrations (MICs) of amphotericin B and lipid-based amphotericin B formulations against isolates of Aspergillus spp. were tested using a broth microdilution method. Twelve isolates of Aspergillus fumigatus, eight of Aspergillus flavus, six of Aspergillus niger and seven of Aspergillus terreus were examined. In addition, an assay for hyphae of Aspergillus spp. was performed since the invasive form is manifested by the appearance of hyphal structures. MICs of hyphae against lipid-based amphotericin B formulations were within three dilutions higher than those against conidia for almost all isolates of Aspergillus spp. (P < 0.01). In contrast, the differences in the in vitro efficacies of amphotericin B were the lowest for Aspergillus spp. This study demonstrates the importance of the type of inoculum used to test antifungal susceptibilities of Aspergillus spp. The significance of these results for in vivo outcome needs to be determined.

Antifungal Properties of 5-hydroxytryptamine (serotonin) Against Candida Species in Vitro

Journal of Medical Microbiology. Feb, 2003  |  Pubmed ID: 12543924

In this study the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) of 5-hydroxytryptamine (5 HT, serotonin) against clinical isolates of Candida albicans (n = 11), Candida glabrata (n = 9), Candida tropicalis (n = 10) and Candida parapsilosis (ATCC 22019) using a broth microdilution test were investigated. In addition, it was examined whether delayed regrowth as a post-antifungal effect results following short exposure to 5 HT. 5 HT showed antifungal activity towards all isolates of Candida spp. The isolates yielded comparable MIC and MFC values of 5 HT in the range 0.91-7.34 mM and 1.83-14.68 mM, respectively. A lag in regrowth was dependent on the concentration tested. Treatment for 3 h at concentrations of 5 HT below and equipotent to the MFC resulted in a delayed regrowth of 8-12 h for isolates of Candida spp. In conclusion, these in vitro studies clearly demonstrate antifungal effects of 5 HT. Identifying the mode of action could be of great help in developing and researching new antifungal drugs.

Interaction of Sertraline with Candida Species Selectively Attenuates Fungal Virulence in Vitro

FEMS Immunology and Medical Microbiology. Jan, 2003  |  Pubmed ID: 12589952

This study investigated whether the interaction between isolates of Candida albicans (n=7), Candida parapsilosis (n=3), Candida krusei (n=2), Candida dubliniensis (n=1) and sertraline, a typical selective serotonin reuptake inhibitor, alters candidal virulence. Sertraline treatment of Candida spp. significantly (P<0.05) affected hyphal elongation, phospholipase activity, production of secreted aspartyl proteinases and fungal viability. In addition, monocyte-derived macrophages (MDMs) treated with sertraline reduced inhibition of blastoconidia germination in comparison to MDMs alone. In conclusion, our findings suggest that the interaction between sertraline and Candida spp. may also diminish the virulence properties of this fungal pathogen in vivo.

Importance of the Terminal Complement Components for Immune Defence Against Candida

International Journal of Medical Microbiology : IJMM. Feb, 2003  |  Pubmed ID: 12635935

Candida activates complement via all three pathways leading to opsonisation and anaphylaxis. The aim of the study was to investigate the influence of the terminal complement system on Candida infections. Thus, fungal cell growth, mitochondrial activity and phagocytosis by polymorphonuclear leukocytes (PMNLs) as well as specific virulence factors, such as release of secreted aspartic protease (Sap) and adherence to epithelial cells, were assessed under the influence of normal or C6/C7-depleted serum. Candida (C.) dubliniensis was used in all experiments as prototype because of its known increased expression of Saps and its strong geno- and phenotypical similarity to the most abundant Candida species C. albicans. Being exposed to sufficient quantities of complement, fungal growth decreased and phagocytosis increased but mitochondrial activities of the yeast increased as well. Concerning the virulence factors, both adhesion and especially Sap release were markedly reduced in the presence of high serum concentrations. Interestingly, at low serum concentrations some opposite effects (an augmented cell growth, a higher Sap release and a stronger adhesion) were observed. In particular, it was shown that the presence of terminal complement factors, and thus the generation of the membrane attack complex, clearly induced a higher fungal mitochondrial activation and has an effect on host defence against yeast cells by augmenting phagocytosis.

Effect of Increasing Inoculum Sizes of Aspergillus Hyphae on MICs and MFCs of Antifungal Agents by Broth Microdilution Method

International Journal of Antimicrobial Agents. Mar, 2003  |  Pubmed ID: 12636983

In order to investigate the influence of different hyphal inoculum sizes on minimal inhibition concentrations (MICs) and minimum fungicidal concentrations (MFCs) of amphotericin B (AMB), voriconazole and itraconazole, five isolates each of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Aspergillus terreus were studied using a broth microdilution method. Three inoculum sizes were used: 1 x 10(3)-5 x 10(3), 1 x 10(4)-5 x 10(4) and 1 x 10(5)-5 x 10(5) cfu/ml. MICs and MFCs were read at 24 and 48 h at 35 degrees C. For all species tested, AMB MICs and MFCs were minimally affected by inoculum size on. However inoculum size significantly affected MICs and MFCs of voriconazole and itraconazole; there was an increase of up to 6-fold in MICs and MFCs for the various aspergilli when the inoculum increased from 10(3) to 10(5) cfu/ml (P<0.05). Thus azoles showed significant inoculum effects, while AMB showed comparatively minimum inoculum effects against Aspergillus spp.

HIV-1 and Its Transmembrane Protein Gp41 Bind to Different Candida Species Modulating Adhesion

FEMS Immunology and Medical Microbiology. Jun, 2003  |  Pubmed ID: 12770763

Oral candidiasis in HIV-1-infected individuals is widely believed to be triggered by the acquired T-lymphocyte immunodeficiency. Recently, binding of the HIV-1 envelope protein gp160 and its subunit gp41, and also of the whole virus itself, to Candida albicans has been shown. The present study shows that, in addition to C. albicans, HIV-1 gp41 also binds to yeast and hyphal forms of Candida dubliniensis, a species which is closely related to C. albicans, and to Candida tropicalis but not to Candida krusei, Candida glabrata or Saccharomyces cerevisiae. The previous finding that gp41 binding to C. albicans augments fungal virulence in vitro is supported by the observation that the yeast showed an enhanced adhesion to HIV-infected H9 cells in comparison to uninfected cells. In line with these results soluble gp41 itself reduced binding of C. albicans to both endothelial and epithelial cell lines, confirming a dominant role of the gp41 binding moiety on the surface of Candida for adhesion. Surface-associated secreted aspartic proteinases (Saps) play an important role in candidial adhesion, but are not likely to be involved in the interaction as gp41 binding to the C. albicans parental wild-type strain was comparable to that of three different isogenic Sap deletion mutants. Furthermore, gp41 binding to the yeast killer toxin-susceptible C. albicans strain 10S was not inhibitable by an anti-YKT receptor antibody. In conclusion, HIV-1 interacts with different clinically important Candida spp., and may thereby affect the outcome of the respective fungal infection.

Diagnosing and Monitoring of Invasive Aspergillosis During Antifungal Therapy by Polymerase Chain Reaction: an Experimental Study in Mice

Diagnostic Microbiology and Infectious Disease. Dec, 2003  |  Pubmed ID: 14711477

This study evaluated the value of polymerase chain reaction (PCR) for diagnosing and monitoring of invasive aspergillosis during amphotericin B therapy. PCR, microscopy and culture of tissues samples (n = 126) and blood samples (n = 78) of experimentally infected mice (n = 42) were performed. The PCR results of treated were compared to those of untreated animals; Aspergillus fumigatus and A. terreus were used in this study. In the amphotericin B treated group the sensitivities of PCR, microscopic examination and culture of the various tissues were 69, 58, and 53%, respectively; the specificities of all examinations were 100%. In the untreated group the sensitivities of PCR, microscopic examination, and culture were 72, 64, and 57%, respectively; the specificities of all examinations were 100%. The 78 blood samples taken from mice under therapy were tested by PCR over a period of 8 days following Aspergillus infection. The test sensitivity was 77%, the specificity 46%, the positive predictive value 59%, and the negative predictive value 67%. In the untreated group the sensitivity was 92%, the specificity 46%, the positive predictive value 63%, and the negative predictive value 86%. The results suggest that this PCR method has possible clinical value for improving the diagnosis of invasive Aspergillus infection. Monitoring of blood under antifungal therapy is not recommended.

HIV-1 Induces Complement Factor C3 Synthesis in Astrocytes and Neurons by Modulation of Promoter Activity

Molecular Immunology. Feb, 2004  |  Pubmed ID: 14725791

Virus-induced complement expression and activation in the brain is hypothesized to contribute to the process of neurodegeneration in AIDS-associated neurological disorders. Previous experiments have shown that the human immunodeficiency virus (HIV) upregulates the low basal production of complement factor C3 in astrocytes and neurons. Since inhibition of complement synthesis and activation in the brain may represent a putative therapeutic goal to prevent virus-induced damage, we analysed the mechanism of the HIV-induced modulation of C3 expression. Detailed studies using different C3 promoter constructs revealed that HIV activates the synthesis of C3 by stimulation of the promoter. This HIV-induced promoter activation could be measured both in different astrocytic cell lines and in neurons. Deletion constructs of the C3 promoter defined the IL-6/IL-1beta responsive element within the promoter region as a central element for the responsiveness of the C3 promoter towards the influence of HIV. A binding site for the transcription factor C/EBPdelta was identified as important regulatory domain within the IL-6/IL-1beta responsive element, since a point mutation which eliminates the binding capacity of C/EBPdelta to this site also abolishes the induction by HIV-1. Similarly, the viral proteins Nef and gp41 which had also been shown to stimulate the synthesis of C3, exert their effect via the IL-6/IL-1beta responsive element with binding of the transcription factor C/EBPdelta representing the critical step. Our experiments clearly define the mechanism for the induction of complement factors in the HIV-infected brain and reveal a decisive role of the regulator protein C/EBPdelta for the HIV-induced increase in C3 expression.

The Role of Complement in Invasive Fungal Infections

Mycoses. Apr, 2004  |  Pubmed ID: 15078425

New therapeutic approaches enable organ transplantations and guarantee longer survival for AIDS patients or patients with haematological neoplasia. The price for these medical advances is immunosuppression and thus enhanced susceptibility to opportunistic fungal infections. As a consequence invasive fungal infections are on the march in modern medicine. Therapeutic limitations and difficulties strongly demand for a deeper understanding of the interaction between the various fungi and the hosts' innate and adaptive immune defence system. This understanding is the essential prerequisite for a potential therapeutic approach, which may support specifically the insufficient antifungal attack of the host. In the present article, we therefore review the current knowledge of the role of the complement system as a central part of innate immunity and as a fine tuner of adaptive immunity in the pathogenesis of invasive fungal infections, such as aspergillosis, candidosis, cryptococcosis, paracoccidioidomycosis, blastomycosis and histoplasmosis.

Complement Synthesis and Activation in the Brain of SIV-infected Monkeys

Journal of Neuroimmunology. Jun, 2004  |  Pubmed ID: 15145603

Complement is one of the most critical defence tools against cerebral infections, but uncontrolled complement biosynthesis and activation can induce profound brain tissue damage. To clarify the role of complement in the pathogenesis of AIDS-associated neurological disorders, we analysed the synthesis of complement in the brains of SIV-infected rhesus macaques. Using immunohistochemical staining we could show that the cerebral synthesis of complement factors C1q and C3 was strongly upregulated in SIV-infected monkeys compared to the spontaneous synthesis in uninfected control monkeys. Astrocytes, neurons, microglia, infiltrating macrophages and multinuclear giant cells all contribute to the high amounts of C1q and C3 in the brain. Secreted C1q and C3 are also deposited on the membrane of neurons, a prerequisite for formation of the membrane-driven lytic membrane attack complex. The membrane deposition thus might suggest complement-induced lysis of bystander neurons as a potential mechanism for cell damage during viral infection of the brain.

Diagnosing Invasive Aspergillosis During Antifungal Therapy by PCR Analysis of Blood Samples

Journal of Clinical Microbiology. Sep, 2004  |  Pubmed ID: 15365004

We evaluated the value of Aspergillus PCR as a tool for diagnosing invasive aspergillosis from whole-blood samples during antifungal therapy. In a 3-year study, 36 patients receiving antifungal therapy due to chest radiographic findings highly suggestive of fungal pneumonia were evaluated. The PCR results from whole-blood samples were compared to those obtained from bronchoalveolar lavage fluids and/or tissue specimens. A total of 205 whole-blood samples, 15 fine-needle aspirations or tissue biopsy specimens, and 21 bronchoalveolar lavage fluids and tracheal secretions were analyzed using PCR. Of the 36 patients, 15 had proven, 9 had probable, and 12 had possible invasive Aspergillus infection according to European Organization for Research and Treatment of Cancer/Mycosis Study Group definitions. For patients with proven infection the sensitivity values of PCR in lung and blood samples were 100 and 40%, respectively. The negative predictive value of blood monitoring under conditions of antifungal treatment was 44%. Clearance of fungal DNA from blood was associated with resolution of clinical symptoms in six of nine patients with proven infection. Repeated positive PCR results for Aspergillus were associated with fatal outcome, as three of six patients died. For patients with probable infection the sensitivity values of PCR in lung fluid and blood were 66 and 44%, respectively. The benefit of PCR diagnosis using whole-blood samples is limited when sampling takes place after treatment has been started. Performance of Aspergillus PCR using tissue samples is recommended in addition to microscopic examination and culture technique for sensitive detection of fungal infection.

Disseminated Infection with Prototheca Zopfii After Unrelated Stem Cell Transplantation for Leukemia

Journal of Clinical Microbiology. Oct, 2004  |  Pubmed ID: 15472379

Disseminated infection with Prototheca zopfii is a rare disease in immunosuppressed patients. We here report the first case of lethal infection with P. zopfii following unrelated stem cell transplantation for leukemia. Breakthrough protothecosis occurred during long-term administration of voriconazole in the case of pulmonary aspergillosis.

Fusarium Verticillioides Abscess of the Nasal Septum in an Immunosuppressed Child: Case Report and Identification of the Morphologically Atypical Fungal Strain

Journal of Clinical Microbiology. Apr, 2005  |  Pubmed ID: 15815043

Morphologically atypical Fusarium verticillioides causing a nasal abscess in a severely immunosuppressed child was successfully treated with repeated surgical intervention and liposomal amphotericin B, despite amphotericin B resistance in vitro. Definitive identification was achieved by sequencing the translation elongation factor alpha gene after ribosomal sequencing proved inadequate.

The Polypore Mushroom Irpex Lacteus, a New Causative Agent of Fungal Infections

Journal of Clinical Microbiology. Apr, 2005  |  Pubmed ID: 15815046

Irpex lacteus, a wood-decaying basidiomycete, was isolated from a pulmonary abscess of an immunosuppressed child. This medical strain was compared morphologically and by sequencing of the ribosomal intergenic spacers with specimens from both culture collections and herbarium desiccated material. The patient was treated successfully with amphotericin B.

Clinical Evaluation of Aspergillus-PCR for Detection of Invasive Aspergillosis in Immunosuppressed Patients

Mycoses. 2005  |  Pubmed ID: 15826281

We evaluated the utility of Aspergillus PCR as a tool in diagnosing invasive aspergillosis in patients at risk. Aspergillosis was assessed according to the European Organization for Research and Treatment of Cancer (EORTC), Mycosis Study Group definitions. Nine and seven patients with proven and probable aspergillosis respectively were evaluated. Whole blood samples prior (n = 41) and during antifungal treatment (n = 67), and tissue specimens (n = 9) and/or bronchoalveolar lavage fluids (n = 7) were investigated. In patients with proven infections, the sensitivities of PCR of lung samples were 100%, of blood samples prior treatment were 66%, and during treatment 55%. Clearance of fungal DNA from blood was associated with resolution of clinical symptoms in two of four patients with proven infection. Consecutive positive PCR results for Aspergillus are fatal as two of five patients died. In patients with probable infections, the sensitivities of PCR of lung fluids were 85%, of blood samples prior treatment were 57%, and during treatment 42%. The benefits of PCR diagnosing and screening of whole blood are limited if sampling takes place once treatment has started. The performance of Aspergillus PCR from tissue samples should be recommended in addition to microscopic examination and culture technique for sensitive detection of fungal infection.

In Vitro Activities of Amphotericin B and Voriconazole Against Aleurioconidia from Aspergillus Terreus

Antimicrobial Agents and Chemotherapy. Jun, 2005  |  Pubmed ID: 15917567

This study used aleurioconidia as inoculum and compared the MICs of amphotericin B and voriconazole to those obtained for conidia of 31 Aspergillus terreus strains. For conidia and aleurioconidia, the MIC at which 90% of strains were inhibited was 2.5 microg/ml and 5 microg/ml with amphotericin B and 1 microg/ml and 2 microg/ml with voriconazole.

Antifungal Activity of the Local Complement System in Cerebral Aspergillosis

Microbes and Infection. Oct, 2005  |  Pubmed ID: 16027023

Dissemination of aspergillosis into the central nervous system is associated with nearly 100% mortality. To study the reasons for the antifungal immune failure we analyzed the efficacy of cerebral complement to combat the fungus Aspergillus. Incubation of Aspergillus in non-inflammatory cerebrospinal fluid (CSF) revealed that complement levels were sufficient to obtain a deposition on the surface, but opsonization was much weaker than in serum. Consequently complement deposition from normal CSF on fungal surface stimulated a very low phagocytic activity of microglia, granulocytes, monocytes and macrophages compared to stimulation by conidia opsonized in serum. Similarly, opsonization of Aspergillus by CSF was not sufficient to induce an oxidative burst in infiltrating granulocytes, whereas conidia opsonized in serum induced a clear respiratory signal. Thus, granulocytes were capable of considerably reducing the viability of serum-opsonized Aspergillus conidia, but not of conidia opsonized in CSF. The limited efficacy of antifungal attack by cerebral complement can be partly compensated by enhanced synthesis, leading to elevated complement concentrations in CSF derived from a patient with cerebral aspergillosis. This inflammatory CSF was able to induce (i) a higher complement deposition on the Aspergillus surface than non-inflammatory CSF, (ii) an accumulation of complement activation products and (iii) an increase in phagocytic and killing activity of infiltrating granulocytes. However, levels and efficacy of the serum-derived complement were not reached. These data indicate that low local complement synthesis and activation may represent a central reason for the insufficient antifungal defense in the brain and the high mortality rate of cerebral aspergillosis.

Tolerability of N-chlorotaurine in Chronic Rhinosinusitis Applied Via Yamik Catheter

Auris, Nasus, Larynx. Dec, 2005  |  Pubmed ID: 16040219

A rational approach in the treatment of chronic rhinosinusitis (CRS) is the intranasal application of antiseptic agents, due to the pathogenetic role of bacteria and fungi. N-Chlorotaurine (NCT), a mild endogenous oxidant with broad-spectrum antimicrobial activity, has been tested for the first time in CRS.

Epidemiology and Outcome of Infections Due to Aspergillus Terreus: 10-year Single Centre Experience

British Journal of Haematology. Oct, 2005  |  Pubmed ID: 16197450

Aspergillus terreus, a less common pathogen, appears to be an emerging cause of infection at our institution, the Medical University Hospital of Innsbruck. Thus the epidemiology and outcome of A. terreus infections over the past 10 years was assessed. We analysed 67 cases of proven invasive aspergillosis (IA) according to the European Organisation for Research and Treatment of Cancer/Mycoses Study Group criteria, investigated antifungal susceptibility of amphotericin B (AMB), voriconazole and caspofungin and performed molecular typing of A. terreus. Patients with proven IA caused by A. terreus (n = 32) and non-A. terreus (n = 35) were evaluated. The two groups were comparable in terms of age, gender, underlying disease, antifungal prophylaxis and duration of neutropenia (P > 0.05). Leukaemia was the most common underlying malignancy. Fungal dissemination occurred in 63% of the patients. Aspergillus terreus infections were associated with a lower response rate to AMB therapy (20%), compared with 47% for patients with non-A. terreus infections (P < 0.05). In vitro, A. terreus was found to be resistant to AMB and molecular typing discriminated between patients isolates, showing a high strain diversity with 26 distinct types (I-XXVI) identified by combination of three primers. Aspergillus terreus infections displayed evidence of AMB resistance in vitro and in vivo and were associated with a high rate of dissemination and poor outcome; A. terreus causes systemic infections of endemic character in Tyrol, Austria. The onset of A. terreus infection depends not on the degree of immunosuppression but on environmental Aspergillus spp. exposure.

Central Venous Catheter Colonization in Critically Ill Patients: a Prospective, Randomized, Controlled Study Comparing Standard with Two Antiseptic-impregnated Catheters

Anesthesia and Analgesia. Dec, 2005  |  Pubmed ID: 16301258

In this prospective, randomized, controlled, unblinded study, we compared colonization rates of a standard, unimpregnated central venous catheter (CVC) with rates for silver-coated and chlorhexidine-silversulfadiazine (CH-SS)-impregnated CVC. Patient characteristics, CVC insertion site, indwelling time, and colonization detected by semiquantitative and quantitative microbiologic techniques were documented. Two-hundred-seventy-five critically ill patients were included into the study protocol. One-hundred-sixty standard, 160 silver (S)-coated, and 165 externally impregnated CH-SS CVC were inserted. There was a significant difference in CVC colonization rates among study groups (P = 0.029). There was no difference in the colonization rate and the colonization per 1000 catheter days between standard and S-coated (P = 0.564; P = 0.24) or CH-SS-coated CVC (P= 0.795; P = 0.639). When comparing antiseptic CVC with each other, colonization rates were significantly less with CH-SS-impregnated than with S-coated CVC (16.9% versus 7.3%; P = 0.01; 18.2 versus 7.5 of 1000 catheter days; P = 0.003; relative risk, 0.43; 95% confidence interval, 0.21-0.85). Whereas standard and S-coated CVC were first colonized 2 and 3 days after insertion, respectively, CH-SS CVC were first colonized only after 7 days. In conclusion, antiseptic-impregnated CVC could not prevent catheter colonization when compared with standard polyurethane catheters in a critical care setting with infrequent catheter colonization rates and CVC left in place for >10 days.

Posaconazole Enhances the Activity of Amphotericin B Against Aspergillus Hyphae in Vitro

Antimicrobial Agents and Chemotherapy. Feb, 2007  |  Pubmed ID: 17116665

The MICs and fractional inhibitory concentrations of posaconazole (POS) and voriconazole (VRZ), alone and in combination with amphotericin B (AMB), for the conidia and hyphae of 100 Aspergillus isolates were evaluated. POS-AMB had more synergistic activity against hyphae (75% of isolates) than VRZ-AMB (37%) and significantly more synergistic activity against hyphae than against conidia (12%).

Interaction of 5-hydroxytryptamine (serotonin) Against Aspergillus Spp. in Vitro

International Journal of Antimicrobial Agents. Apr, 2007  |  Pubmed ID: 17276041

This study examined the direct interaction of serotonin (5-hydroxytryptamine (5-HT)) with Aspergillus species. Accumulation of 5-HT in aspergilli was investigated by immunofluorescence staining and laser confocal scanning microscopy. The influence of 5-HT on fungal ergosterol content, cell membrane integrity, fungal growth and hyphal elongation was determined. 5-HT was localised in the cytoplasm of Aspergillus spp., as 5-HT fluorescent signals appeared after 30min at 4 degrees C and in the presence of inhibitors of oxidative phosphorylation. 5-HT treatment of Aspergillus spp. significantly affected ergosterol synthesis, fungal cell membrane integrity and hyphal elongation (P<0.05). 5-HT treatment for 4h resulted in a lag of re-growth (post-antifungal effect). In conclusion, our findings suggest that 5-HT affects hyphal growth and diminishes fungal cell membrane integrity.

Human Protothecosis

Clinical Microbiology Reviews. Apr, 2007  |  Pubmed ID: 17428884

Human protothecosis is a rare infection caused by members of the genus Prototheca. Prototheca species are generally considered to be achlorophyllic algae and are ubiquitous in nature. The occurrence of protothecosis can be local or disseminated and acute or chronic, with the latter being more common. Diseases have been classified as (i) cutaneous lesions, (ii) olecranon bursitis, or (iii) disseminated or systemic manifestations. Infections can occur in both immunocompetent and immunosuppressed patients, although more severe and disseminated infections tend to occur in immunocompromised individuals. Prototheca wickerhamii and Prototheca zopfii have been associated with human disease. Usually, treatment involves medical and surgical approaches; treatment failure is not uncommon. Antifungals such as ketoconazole, itraconazole, fluconazole, and amphotericin B are the most commonly used drugs to date. Among them, amphotericin B displays the best activity against Prototheca spp. Diagnosis is largely made upon detection of characteristic structures observed on histopathologic examination of tissue.

In Vitro Determination of Phagocytosis and Intracellular Killing of Aspergillus Species by Mononuclear Phagocytes

Mycopathologia. Jun, 2007  |  Pubmed ID: 17458710

We investigated phagocytosis and intracellular killing of clinical and environmental isolates of Aspergillus spp. by human monocyte-derived macrophages (MDMs). Serial pathogens such as Aspergillus fumigatus, Aspergillus flavus and Aspergillus terreus were examined with a microbiological assay. Phagocytosis for resting conidia of Aspergillus spp. was similar for all isolates tested. During 30 min of incubation phagocytosis ranged from 49.9% to 85.5% for clinical isolates and from 40.3% to 87.1% for environmental isolates. MDMs killed A. fumigatus, A. flavus and A. terreus conidia after ingestion for 120 min, as shown by a decrease in colony forming units (cfu) count of intracellular fungi. The killing index for all isolates of Aspergillus spp., ranged from 12.1 +/- 1.1% to 90.3 +/- 10.4%; isolate-dependent (P < 0.01) differences against the fungicidal action of MDMs were observed. In conclusion, significant differences were noted for killing indices between several strains of Aspergillus spp. whereas phagocytosis was similar for all isolates tested in vitro. No differences were observed within environmental and clinical isolates.

Absent Reduction by HIV Protease Inhibitors of Candida Albicans Adhesion to Endothelial Cells

Mycoses. May, 2007  |  Pubmed ID: 17472611

Highly active antiretroviral therapy including HIV protease inhibitors has led to a marked reduction of clinically relevant mucosal candidiasis. We have previously shown that HIV protease inhibitors directly inhibit adhesion of Candida albicans to epithelial cells at concentrations that are reached in vivo during antiretroviral therapy. The aim of this study was to establish whether HIV protease inhibitors also inhibit adhesion of Candida to endothelial cells, which play a major role in systemic fungal disease. Three C. albicans strains were incubated with human umbilical vein endothelial cells or an endothelial cell line in the presence of either Ritonavir, Saquinavir or Indinavir. Subsequently, adherence was determined by counting colony-forming units. The results were comparable and revealed that Ritonavir and Saquinavir significantly inhibited adherence to endothelial cells at only very high concentrations which are likely not reached in vivo, and Indinavir did not even inhibit then. Inhibition of adhesion of C. albicans to human cells by HIV protease inhibitors is not a general feature, but strongly cell type-dependent, and clearly not observed for endothelial cells in vitro, which are a main target of systemic candidiasis in vivo.

Prevention of Transfusion of Platelet Components Contaminated with Low Levels of Bacteria: a Comparison of Bacteria Culture and Pathogen Inactivation Methods

Transfusion. Jul, 2007  |  Pubmed ID: 17581146

This study compared the efficacy of bacterial detection with inactivation for reducing the risk associated with transfusion of platelet (PLT) components contaminated with low levels of bacteria.

Molecular Typing of Aspergillus Terreus Isolates Collected in Houston, Texas, and Innsbruck, Austria: Evidence of Great Genetic Diversity

Journal of Clinical Microbiology. Aug, 2007  |  Pubmed ID: 17581930

Aspergillus terreus isolates collected from patients at The M. D. Anderson Cancer Center in Houston, TX, and at The University Hospital of Innsbruck, Austria, were analyzed using random amplification of polymorphic DNA-PCR with three different primers. No strain similarity in either institution was detected, indicating great genetic diversity of A. terreus.

Pseudallescheria Boydii Infection After Liver Retransplantation

Liver Transplantation : Official Publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society. Jul, 2007  |  Pubmed ID: 17600358

The Value of Computed Tomography-guided Percutaneous Lung Biopsy for Diagnosis of Invasive Fungal Infection in Immunocompromised Patients

Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America. Oct, 2007  |  Pubmed ID: 17806041

We assessed Calcofluor white staining, Aspergillus polymerase chain reaction, and a galactomannan enzyme immunoassay for diagnosis of fungal infection with use of computed tomography-guided percutaneous lung biopsy specimens obtained from 61 patients. The sensitivity and specificity of computerized tomography, Aspergillus polymerase chain reaction, and galactomannan enzyme immunoassay were 100% and 50%, 100% and 86%, and 88% and 94%, respectively.

Bacterial Contamination of Solutions for Parenteral Administration for Single- and Multiple-dose Vials After Multiple Use in the Hospital

Wiener Medizinische Wochenschrift (1946). 2007  |  Pubmed ID: 17922089

Outbreaks traced to bacterial contamination of multiple-dose vials are reported in the literature. During a four-month period, multi-dose vials (MDVs), single-dose vials (SDVs), and vials containing self-prepared admixtures were collected from various wards to analyse sterility of their contents. We examined 68 commercially available MDVs containing sodium chloride 0.9% or heparin with added preservative and 17 single dose vials (SDVs) containing aqua ad injectionem or sodium chloride 0.9% and 11 vials with admixtures (ADX) of heparin and sodium chloride 0.9%, both without preservative. Four of 96 (4.17%) vials were not sterile: two of them were contaminated with spore-forming bacteria, two with coagulase negative Staphylococci. Three of the samples were MDVs containing a preservative. The date of the first use was not marked on 28% of the vials. Twenty-eight samples were multiply used, although they were SDVs or ADXs without preservative or without an adequate amount of preservative, respectively. In 15 of 68 MDVs, the time limit after the first use was exceeded. On average, the volume of the samples was 80% of the original volume. A proportion of 4% of vials was not sterile. A training programme for health care workers in aseptic techniques and for validation of the preparation of solutions for parenteral use should be installed.

Alternaria Alternata Soft Tissue Infection in a Forearm Transplant Recipient

Surgical Infections. Oct, 2007  |  Pubmed ID: 17999589

Composite tissue allograft recipients require intensive immunosuppression and are therefore at risk of infections. Filamentous fungal infections are among the complications most difficult to manage in organ transplant recipients.

[Serological Tests for Diagnosing Fungal Infections]

Wiener Medizinische Wochenschrift (1946). 2007  |  Pubmed ID: 18030559

Considerable interest has been made in developing nonculture approaches to diagnosing fungal infections, as early diagnosis may guide appropriate treatment and prevent mortality. These approaches include detection of specific host immune responses to fungal antigens, and detection of specific macromolecular antigens using immunologic reagents. The detection of a specific host antibody response is attractive because such tests can be performed rapidly and do not require invasive sampling procedures. However, presence of host antibodies does not always correlate with presence of invasive disease, especially in patients whose abilities to produce specific immunoglobulin responses may be impeded by immunosuppressive drugs and/or serious underlying diseases. Detection of macromolecular microbial antigens generally requires a relatively large microbial burden, which may limit assay sensitivity.

Standard Dosing Regimen of Liposomal Amphotericin B is As Effective As a High-loading Dose for Patients with Invasive Aspergillosis: AmBiLoad Trial

Expert Review of Anti-infective Therapy. Dec, 2007  |  Pubmed ID: 18039077

Invasive mold infections continue to account for significant morbidity and mortality in immunocompromised patients; outcomes are dependent on both underlying host factors and appropriate therapy. The antifungal armamentarium has gradually increased during the past, with liposomal amphotericin B (L-AMB) being an important representative. Still, the question of what dose to use - a maximum tolerated or a minimum effective - has yet to be answered. On this basis, a randomized trial comparing a high-loading dose regimen with a standard dosing of L-AMB (AmBiLoad trial) for primary therapy of mold infections was initiated. No significant differences in response between the treatment groups were detected, although recipients of the 10-mg/kg daily dose experienced higher rates of nephrotoxicity and hypokalemia. Uncontrolled malignancy and allogeneic stem cell transplantation were significantly associated with poor survival. This article analyzes the study, discusses the rationale and the results and concludes that this study supports the routine application of L-AMB.

Gliotoxin Production by Clinical and Environmental Aspergillus Fumigatus Strains

International Journal of Medical Microbiology : IJMM. Apr, 2008  |  Pubmed ID: 17574915

The mycotoxin gliotoxin is produced by fungi of the genus Aspergillus, including the important human pathogen Aspergillus fumigatus. Gliotoxin exerts a broad spectrum of immunosuppressive effects in vitro and is detectable in the sera of patients suffering from invasive aspergillosis. In order to correlate the pathogenic potential of A. fumigatus with the ability to produce gliotoxin and to investigate the taxonomic distribution of gliotoxin-producing Aspergillus strains among clinical isolates, a total of 158 Aspergillus isolates comprising four different species (A. fumigatus, n=100; A. terreus, n=27; A. niger, n=16; A. flavus, n=15) were collected from different medical centers (some originating from probable cases of aspergillosis) and from environmental samples in Germany and Austria. Remarkably, gliotoxin was detected in most culture filtrates of A. fumigatus of both clinical (98%) and environmental (96%) origin. The toxin was also detected, with decreasing frequency, in culture filtrates of A. niger (56%), A. terreus (37%), and A. flavus (13%). The highest gliotoxin concentrations were detected in A. fumigatus strains of clinical (max. 21.35 microg/ml, mean 5.75 microg/ml) and environmental (max. 26.25 microg/ml, mean 5.27 microg/ml) origin. Gliotoxin productivity of other Aspergillus species was significantly lower. Culture supernatants of A. fumigatus strains lacking gliotoxin production showed a significantly lower cytotoxicity on macrophage-like cells and T-cells in vitro. In contrast, lack of gliotoxin production in the other Aspergillus species tested had no significant influence on the cytotoxic effect of culture supernatant on these immune cells.

Silver Segregation and Bacterial Growth of Intraventricular Catheters Impregnated with Silver Nanoparticles in Cerebrospinal Fluid Drainages

Neurological Research. Apr, 2008  |  Pubmed ID: 17767809

Intraventricular catheters impregnated with silver nanoparticles are developed to reduce catheter-associated infections in cerebrospinal fluid (CSF) drainages. Silver released from these new catheters should have an anti-microbacterial effect. This study examines the silver ion release and a potential effect of bacterial growth in an in vitro experiment.

Genotyping of Paracoccidioides Brasiliensis Directly from Paraffin Embedded Tissue

Medical Mycology. Feb, 2008  |  Pubmed ID: 17885941

Based on polymorphisms of the gp43 precursor gene, genotyping of Paracoccidioides brasiliensis was achieved in 6 out of 10 paraffin-embedded tissue samples by modifying a nested PCR procedure used in the diagnosis of the fungal infection. Nine of the samples originated in Brazil. Three sequences were identical to a previously published consensus sequence identifying the P. brasiliensis isolates as members of the formerly named species 1. In contrast, two sequences revealed substitutions identical to isolates of the phylogenetic species 2. Applying the method to a lung biopsy from a proven case of paracoccidioidomycosis diagnosed in Austria, the gp43 sequence revealed substitutions so far only described in five strains originating from Venezuela. Combining travel history and result of this new method, the most probable country of origin of the infections could be identified. Since endemic mycosis are often diagnosed by histopathology only, our method could help to extend epidemiological studies of paracoccidioidomycosis.

Potential Basis for Amphotericin B Resistance in Aspergillus Terreus

Antimicrobial Agents and Chemotherapy. Apr, 2008  |  Pubmed ID: 18268082

This study investigated the basis for intrinsic amphotericin B (AMB) resistance in Aspergillus terreus. The ergosterol content, cell wall composition, and lipid peroxidation level had no influence on AMB resistance. The level of catalase production in A. terreus was significantly higher than that in A. fumigatus (P < 0.05). This higher-level production may contribute to AMB resistance in A. terreus since oxidative damage has been implicated in AMB action.

Susceptibility Testing of Anidulafungin and Voriconazole Alone and in Combination Against Conidia and Hyphae of Aspergillus Spp. Under Hypoxic Conditions

Antimicrobial Agents and Chemotherapy. May, 2008  |  Pubmed ID: 18347112

MICs and fractional inhibitory concentrations were evaluated for anidulafungin and voriconazole alone and in combination against conidia and hyphae under hypoxic (1% oxygen-5% CO(2)-94% nitrogen) conditions against 31 Aspergillus isolates. Anidulafungin exhibited excellent activity against conidia and hyphae of Aspergillus spp. The visual reading of the MIC for anidulafungin was optimal under hypoxic conditions.

Basidiomycete Metabolites Attenuate Virulence Properties of Candida Albicans in Vitro

Mycoses. Nov, 2008  |  Pubmed ID: 18422912

Secreted aspartic proteases (Saps) represent an important virulence factor facilitating fungal adherence. Several protease inhibitors (PIs), including HIV PIs, have been shown to reduce Candida adhesion. The aim of this study was to ascertain whether or not the recently discovered PIs Aureoquinone and Laccaridiones A and B, isolated from Basidiomycete cultures, or Bestatin, act as Sap-inhibitors and/or inhibitors of fungal adhesion. Drug effects on candidial Sap-production were determined by Sap-ELISA. Control tubes, in the absence of drug, served as positive controls, while tubes excluding both drug and proteinase induction medium were used as negative controls. Aureoquinone as well as Laccaridiones A and B, but not Bestatin, significantly inhibited Candida albicans adhesion to both epithelial and endothelial cells in a dose dependent manner and also reduced Sap-release (effects were not because of a direct interaction of the Basidiomycete metabolites with secreted Saps). Laccaridione B was consistently found to be the most effective PI tested. Interestingly, these drugs are neither fungistatic nor fungicidal at the concentrations applied. Laccaridione B may represent a promising novel type of antimycotic drug--targeting virulence factors without killing the yeast.

In Vitro Susceptibility-testing in Aspergillus Species

Mycoses. Sep, 2008  |  Pubmed ID: 18422914

Aspergillus species are the most common causes of invasive mould infections in immunocompromised patients. The introduction of new antifungal agents and recent reports of resistance emerging during treatment of Aspergillus infections have highlighted the need for in vitro susceptibility-testing. Various testing procedures have been proposed, including macrodilution and microdilution, agar diffusion, disc diffusion and Etest. At present, one of the most widely used assays is the M38-A reference method for filamentous fungi, published by the Clinical Laboratory Standard Institute and the Etest. Recently, the European Committee on Antimicrobial Susceptibility-testing (EUCAST) has charged its Antifungal Susceptibility-testing Subcommittee (AFST-EUCAST) with the preparation of new guidelines for in vitro susceptibility-testing of antifungals against Aspergillus spp. (EUCAST-AFST-ASPERGILLUS) defining breakpoints. This paper reviews the available methods for antifungal susceptibility-testing in Aspergillus spp. as well as the scant data regarding the clinical implications of in vitro testing.

Posaconazole Enhances the Activity of Amphotericin B Against Hyphae of Zygomycetes in Vitro

Antimicrobial Agents and Chemotherapy. Jul, 2008  |  Pubmed ID: 18458135

The in vitro activity of posaconazole plus amphotericin B against conidia and hyphae of 30 clinical zygomycetes was investigated. The combination of posaconazole with amphotericin B was found to be significantly more synergistic (40%) against hyphae (P < 0.05) than against conidia (10%). Antagonism was not observed.

Establishing in Vitro-in Vivo Correlations for Aspergillus Fumigatus: the Challenge of Azoles Versus Echinocandins

Antimicrobial Agents and Chemotherapy. Oct, 2008  |  Pubmed ID: 18644959

Two clinical isolates of Aspergillus fumigatus, designated AT and DK, were recently obtained from patients failing caspofungin and itraconazole therapy, respectively. The isolates were tested by microdilution for susceptibility to itraconazole, voriconazole, posaconazole, ravuconazole, and caspofungin and by Etest for susceptibility to amphotericin B and caspofungin. Susceptibility testing documented that the DK isolate was azole resistant (itraconazole and posaconazole MICs, >4 microg/ml; voriconazole MIC, 2 microg/ml; ravuconazole MIC, 4 microg/ml), and the resistance was confirmed in a hematogenous mouse model, with mortality and the galactomannan index as the primary and secondary end points. Sequencing of the cyp51A gene revealed the M220K mutation, conferring multiazole resistance. The Etest, but not microdilution, suggested that the AT isolate was resistant to caspofungin (MIC, >32 microg/ml). In the animal model, this isolate showed reduced susceptibility to caspofungin. Sequencing of the FKS1 gene revealed no mutations; the enzyme retained full sensitivity in vitro; and investigation of the polysaccharide composition showed that the beta-(1,3)-glucan proportion was unchanged. However, gene expression profiling by Northern blotting and real-time PCR demonstrated that the FKS gene was expressed at a higher level in the AT isolate than in the susceptible control isolate. To our knowledge, this is the first report to document the presence of multiazole-resistant clinical isolates in Denmark and to demonstrate reduced susceptibility to caspofungin in a clinical A. fumigatus isolate with increased expression of the FKS gene. Further research to determine the prevalence of resistance in A. fumigatus worldwide, and to develop easier and reliable tools for the identification of such isolates in routine laboratories, is warranted.

First Case of Breakthrough Pulmonary Aspergillus Niveus Infection in a Patient After Allogeneic Hematopoietic Stem Cell Transplantation

Diagnostic Microbiology and Infectious Disease. Nov, 2008  |  Pubmed ID: 18687556

Aspergillus niveus is a species uncommon in clinical samples, and to date, invasive fungal infections caused by this fungal pathogen have not been described. This is the 1st report on a pulmonary breakthrough aspergillosis caused by A. niveus in a 21-year-old woman after allogeneic hematopoietic stem cell transplantation for Fanconi anemia.

Activities of Antifungal Agents Against Yeasts and Filamentous Fungi: Assessment According to the Methodology of the European Committee on Antimicrobial Susceptibility Testing

Antimicrobial Agents and Chemotherapy. Oct, 2008  |  Pubmed ID: 18694949

We compared the activities of antifungal agents against a wide range of yeasts and filamentous fungi. The methodology of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for yeasts and spore-forming molds was applied; and a total of 349 clinical isolates of Candida spp., other yeast species, Aspergillus spp., and nondermatophyte non-Aspergillus spp. were investigated. The average geometric mean (GM) of the MICs of the various drugs for Candida spp. were as follows: amphotericin B (AMB), 0.55 microg/ml; liposomal amphotericin B (l-AMB); 0.35 microg/ml; itraconazole (ITC), 0.56 microg/ml; voriconazole (VRC), 0.45 microg/ml; posaconazole (POS), 0.44 microg/ml; and caspofungin (CPF), 0.45 microg/ml. The data indicated that the majority of Candida spp. were susceptible to the traditional and new antifungal drugs. For Aspergillus spp., the average GM MICs of AMB, l-AMB, ITC, VRC, POS, and CPF were 1.49 microg/ml, 1.44 microg/ml, 0.65 microg/ml, 0.34 microg/ml, 0.25 microg/ml, and 0.32 microg/ml, respectively. For the various zygomycetes, the average GM MICs of AMB, l-AMB, ITC, and POS were 1.36 microg/ml, 1.42 microg/ml, 4.37 microg/ml, and 1.65 microg/ml, respectively. Other yeastlike fungi and molds displayed various patterns of susceptibility. In general, the minimal fungicidal concentrations were 1 to 3 dilutions higher than the corresponding MICs. POS, AMB, and l-AMB showed activities against a broader range of fungi than ITC, VRC, and CPF did. Emerging pathogens such as Saccharomyces cerevisiae and Fusarium solani were not killed by any drug. In summary, the EUCAST data showed that the in vitro susceptibilities of yeasts and filamentous fungi are variable, that susceptibility occurs among and within various genera and species, and that susceptibility depends on the antifungal drug tested. AMB, l-AMB, and POS were active against the majority of pathogens, including species that cause rare and difficult-to-treat infections.

Complement and Fungal Pathogens: an Update

Mycoses. Nov, 2008  |  Pubmed ID: 18705662

Fungal infections are a serious complication in immunocompromised patients such as human immunodeficiency virus-infected individuals, patients with organ transplantations or with haematological neoplasia. The lethality of opportunistic fungal infection is high despite a growing arsenal of antimycotic drugs, implying the urgent need for supportive immunological therapies to strengthen the current inefficient antimicrobial defences of the immunocompromised host. Therefore, increasing effort has been directed to investigating the interplay between fungi and the host immunity and thus to find starting points for additional therapeutic approaches. In this article, we review the actual state of the art concerning the role of complement in the pathogenesis of fungal infections. Important aspects include the activation of the complement system by the fungal pathogen, the efficiency of the complement-associated antimicrobial functions and the arsenal of immune evasion strategies applied by the fungi. The twin functions of complement as an interactive player of the innate immunity and at the same time as a modulator of the adaptive immunity make this defence weapon a particularly interesting therapeutic candidate to mobilise a more effective immune response and to strengthen in one fell swoop a broad spectrum of different immune reactions. However, we also mention the 'Yin-Yang' nature of the complement system in fungal infections, as growing evidence assigns to complement a contributory part in the pathogenesis of fungus-induced allergic manifestations.

Human Platelets Attenuate Aspergillus Species Via Granule-dependent Mechanisms

The Journal of Infectious Diseases. Oct, 2008  |  Pubmed ID: 18752432

Using laser scanning microscopy, we investigated whether platelets are capable of internalizing Aspergillus conidia and examined Aspergillus-platelet adherence. The influence of platelets on fungal growth was evaluated by assessing galactomannan (GM) release, hyphal elongation, and colony size. A secretion assay with [(3)H]-serotonin (5-hydroxytryptamine [5-HT]) was performed. Exposure to platelets resulted in significantly decreased GM release (p<.05), hyphal elongation (p<.001), colony size, pigmentation, and 5-HT release ( p<.05). A lack of antifungal effects was observed with the microfilament inhibitor cytochalasin D. Platelets attenuate the virulence of Aspergillus species in vitro on the basis of granule-dependent effects.

Complement Induction and Complement Evasion in Patients with Cerebral Aspergillosis

Microbes and Infection. Nov-Dec, 2008  |  Pubmed ID: 18977454

Cerebral aspergillosis is a mostly lethal infection of the central nervous system. Former results identified low cerebral complement levels as one cause for insufficient immune reaction. Therefore we studied cerebral complement expression after fungal invasion and investigated putative mechanisms of Aspergillus spp to cope with the complement-induced selection pressure. Brain tissue derived from patients with cerebral aspergillosis or non-infected individuals was analyzed immunohistochemically for complement synthesis. Correlations between expression levels were determined statistically. Increased complement synthesis, a prerequisite for strengthened antifungal potency, was visible in resident astrocytes, neurons, oligodendrocytes as well as in infiltrating macrophages after fungal infection. Surprisingly, microglia, although regarded as major immune cells, only marginally participated in synthesis of most complement proteins. Several evasion mechanisms were found that help the fungus to establish a cerebral infection even in the presence of complement: Fungal hyphae limit the surface deposition of C3 and thus interfere with complement-dependent phagocytosis. Furthermore, the "sealing off" in brain abscesses not only inhibits fungal spreading but also forms protection shields against complement attack. Complement indeed seems to represent an important selection pressure and evokes the development of fungal evasion mechanisms. Counteractions for these evasion processes might represent interesting therapeutic approaches.

Significant Alterations in the Epidemiology and Treatment Outcome of Invasive Fungal Infections in Patients with Hematological Malignancies

International Journal of Hematology. Dec, 2008  |  Pubmed ID: 18982251

Invasive fungal infections (IFI) remain a leading cause of morbidity and mortality in immunocompromised patients. This retrospective single-center study analyzed incidence, treatment and outcome of invasive fungal infections in 1,095 patients with hematological malignancies receiving either cytoreductive chemotherapy or autologous or allogeneic hematopoietic stem cell transplantation at our institution between 1995 and 2004. IFI occurred in 167/1,095 (15%) patients with a significant increase over time (12.7% between 1995 and 2000 vs. 18.1% in the later IFI cohort, P = 0.0134). Fifty-four (32%) patients had proven, 70 (42%) patients had probable, and 43 (26%) patients suffered from possible IFI according to EORTC/MSG criteria. In 108/124 (87%) cases with proven or probable IFI, moulds were the causative pathogens. Both, Aspergillus fumigatus (n = 46) and Aspergillus terreus (n = 41) were predominant. Yeast infections (Candida spp.) were documented in 16/124 (10%) cases with proven or probable IFI. Median overall survival of the entire IFI cohort was 7 (3-17) months. Overall survival was significantly better in patients with probable or possible IFI (37 and 38%, respectively) compared with patients with proven IFI (28%, P = 0.019). In 35% of patients, IFI was the principal cause of death with a significant decrease over time (44% in time cohort 1995-2000 vs. 28% in the later IFI cohort, P = 0.018) accompanied by an increased use of novel antifungals. By multivariate analysis, only proven IFI was significantly predictive for death (HR 1.7, P = 0.018). A significant decrease in fungus-related deaths was observed despite a significant increase of IFI over time, probably due to improved diagnostic and therapeutic approaches.

In Vitro Activities of Various Antifungal Drugs Against Aspergillus Terreus: Global Assessment Using the Methodology of the European Committee on Antimicrobial Susceptibility Testing

Antimicrobial Agents and Chemotherapy. Feb, 2009  |  Pubmed ID: 19064891

This study presents in vitro susceptibility data for clinical (n = 48) and environmental (n = 31) isolates of Aspergillus terreus against nine antifungal agents. The methodology of the European Committee on Antimicrobial Susceptibility Testing was applied. Posaconazole and anidulafungin had the lowest and amphotericin B the highest MICs. No differences in susceptibility patterns were observed between environmental and clinical isolates.

Breakthrough Aspergillus Fumigatus and Candida Albicans Double Infection During Caspofungin Treatment: Laboratory Characteristics and Implication for Susceptibility Testing

Antimicrobial Agents and Chemotherapy. Mar, 2009  |  Pubmed ID: 19104024

Caspofungin is used for the treatment of acute invasive candidiasis and as salvage treatment for invasive aspergillosis. We report characteristics of isolates of Candida albicans and Aspergillus fumigatus detected in a patient with breakthrough infection complicating severe gastrointestinal surgery and evaluate the capability of susceptibility methods to identify candin resistance. The susceptibility of C. albicans to caspofungin and anidulafungin was investigated by Etest, microdilution (European Committee on Antibiotic Susceptibility Testing [EUCAST] and CLSI), disk diffusion, agar dilution, and FKS1 sequencing and in a mouse model. Tissue was examined by immunohistochemistry, PCR, and sequencing for the presence of A. fumigatus and resistance mutations. The MICs for the C. albicans isolate were as follows: >32 microg/ml caspofungin and 0.5 microg/ml anidulafungin by Etest, 2 microg/ml caspofungin and 0.125 microg/ml anidulafungin by EUCAST methods, and 1 microg/ml caspofungin and 0.5 microg/ml anidulafungin by CLSI methods. Sequencing of the FKS1 gene revealed a mutation leading to an S645P substitution. Caspofungin and anidulafungin failed to reduce kidney CFU counts in animals inoculated with this isolate (P > 0.05 compared to untreated control animals), while both candins completely sterilized the kidneys in animals infected with a control isolate. Disk diffusion and agar dilution methods clearly separated the two isolates. Immunohistochemistry and sequencing confirmed the presence of A. fumigatus without FSK1 resistance mutations in liver and lung tissues. Breakthrough disseminated aspergillosis and candidiasis developed despite an absence of characteristic FKS1 resistance mutations in the Aspergillus isolates. EUCAST and CLSI methodology did not separate the candin-resistant clinical isolate from the sensitive control isolate as well as did the Etest and agar methods.

Triazole-resistant Candidaemia Following Posaconazole Exposure

International Journal of Antimicrobial Agents. May, 2009  |  Pubmed ID: 19153033

In Vitro Activity of Isavuconazole Against Aspergillus Species and Zygomycetes According to the Methodology of the European Committee on Antimicrobial Susceptibility Testing

Antimicrobial Agents and Chemotherapy. Apr, 2009  |  Pubmed ID: 19164153

We evaluated the MICs of isavuconazole (ISAV) against 96 isolates of Aspergillus species and 36 zygomycetes according to the methodology of the European Committee on Antimicrobial Susceptibility Testing. In addition, the in vitro activity was obtained for hyphal inocula. ISAV exhibited good antifungal activity against the tested isolates with the exception of Aspergillus niger and Mucorales. The in vitro activity of ISAV was comparable to that of voriconazole aside from Mucorales.

Aspergillus Alabamensis, a New Clinically Relevant Species in the Section Terrei

Eukaryotic Cell. May, 2009  |  Pubmed ID: 19304950

Phylogenetic analyses of sequences generated from portions of three genes coding for the proteins enolase (enoA), beta-tubulin (benA), and calmodulin (calM) of a large number of isolates within the section Terrei, genus Aspergillus, revealed the presence of a new cryptic species within this section, Aspergillus alabamensis. Most members of this new cryptic species were recovered as colonizing isolates from immunocompetent patient populations, had decreased in vitro susceptibilities to the antifungal drug amphotericin B, and were morphologically similar to but genetically distinct from Aspergillus terreus isolates.

The Changing Face of Epidemiology of Invasive Fungal Disease in Europe

Mycoses. May, 2009  |  Pubmed ID: 19391253

Invasive fungal diseases (IFDs) are an increasingly common complication in critically ill patients in Europe and are frequently fatal. Because of changes in treatment strategies and the increased use of antifungal prophylaxis, the epidemiology of IFDs has changed substantially in recent years and infections due to Candida species are no longer the majority in many institutions. In contrast, the emergence of non-Candida IFDs such as aspergillosis, zygomycosis and fusariosis has increased. European surveys indicate that Candida albicans is responsible for more than half the cases of invasive candidaemia; however, the occurrence of non-albicans-related IFDs appears to be increasing. Rates of IFD-related mortality in Europe depend on the pathogen, geographical location and underlying patient characteristics, with rates ranging from 28 to 59% for Candida infections and from 38 to 80% for invasive aspergillosis. Early initiation of antifungal therapy is critical for improving outcomes; however, this is complicated by the difficulty in diagnosing IFDs rapidly and accurately. The introduction of new extended-spectrum azole antifungal agents (e.g. voriconazole, posaconazole) and echinocandins (e.g. micafungin, caspofungin, anidulafungin) has increased the number of therapeutic options for early therapy. Choice between agents should be based on a variety of factors, including spectrum of activity, adverse events, drug interactions, route of administration, clinical efficacy of individual agents and local epidemiology.

Association of Fluconazole Pharmacodynamics with Mortality in Patients with Candidemia

Antimicrobial Agents and Chemotherapy. Jun, 2009  |  Pubmed ID: 19458347

Infections Caused by Candida Krusei in Five Transplant and Two Surgical Patients

Surgical Infections. Jun, 2009  |  Pubmed ID: 19485782

Candida krusei (Ck) may cause severe infections in immunocompromised hosts and is innately resistant to fluconazole.

Anidulafungin and Voriconazole in Invasive Fungal Disease: Pharmacological Data and Their Use in Combination

Expert Opinion on Investigational Drugs. Sep, 2009  |  Pubmed ID: 19656074

The incidence of invasive fungal infections has been increasing since the 1980s due to a growing population of immunocompromised and critically ill patients with associated risk factors including immunosuppressive chemotherapy, prolonged periods on intensive care units and infection with HIV. Persons who are severely immunocompromised are particularly vulnerable to infection from molds and yeasts that are often found naturally in the environment. In recent years, several new systemic antifungal agents have been released, significantly increasing options for the treatment of the most serious fungal infections. Newly available drugs as those in the echinocandin class include caspofungin, micafungin and anidulafungin, as well as the newer generation triazoles, voriconazole and posaconazole. In this review, the in vitro and in vivo activity of anidulafungin and voriconazole, both new antimycotic substances with a different mode of action, are analyzed.

Zygomycosis in Solid Organ Transplant Recipients: a Prospective, Matched Case-control Study to Assess Risks for Disease and Outcome

The Journal of Infectious Diseases. Sep, 2009  |  Pubmed ID: 19659439

Clinical characteristics, risks, and outcomes in solid organ transplant (SOT) recipients with zygomycosis in the era of modern immunosuppressive and newer antifungal agent use have not been defined.

Potential Antifungal Effects of Human Platelets Against Zygomycetes in Vitro

The Journal of Infectious Diseases. Oct, 2009  |  Pubmed ID: 19698079

Zygomycosis is increasingly recognized in immunocompromised hosts. We investigated whether platelets become activated after contact with Zygomycetes and adhere to conidial and hyphal structures using immunofluorescence. The platelets' influence on fungal viability was evaluated by assessing hyphal elongation and hyphal damage. Platelets became activated and strongly adhered to conidia and hyphae of Zygomycetes. Platelets induced time dependent damage to hyphae and significantly reduced (P<.05) hyphal elongation. We found that platelets possess antifungal capacities against Zygomycetes based on granule dependent mechanisms and significantly reduce fungal growth and spread, both of which are of major importance in evolving invasive disease.

Diagnosing Invasive Fungal Diseases - Limitations of Microbiological Diagnostic Methods

Expert Opinion on Medical Diagnostics. Jul, 2009  |  Pubmed ID: 23485213

Invasive fungal infections (IFD) are a principal cause of morbidity and mortality in immunosuppressed patients, with case fatality rates ranging from 30 - 80% in neutropenic patients. This results at least partly from difficulties in obtaining a reliable and early diagnosis. Conventional methods, for example culture-based methods, may be difficult and are often delayed. Serological tests such as the galactomannan enzyme immunoassay are at present most promising for diagnosing invasive aspergillosis; however, sensitivity and specificity vary within the studies. Similar data are shown for Aspergillus detection by polymerase chain reaction.

Echinocandin Susceptibility Testing of Candida Species: Comparison of EUCAST EDef 7.1, CLSI M27-A3, Etest, Disk Diffusion, and Agar Dilution Methods with RPMI and Isosensitest Media

Antimicrobial Agents and Chemotherapy. Jan, 2010  |  Pubmed ID: 19884370

This study compared nine susceptibility testing methods and 12 endpoints for anidulafungin, caspofungin, and micafungin with the same collection of blinded FKS hot spot mutant (n = 29) and wild-type isolates (n = 94). The susceptibility tests included EUCAST Edef 7.1, agar dilution, Etest, and disk diffusion with RPMI-1640 plus 2% glucose (2G) and IsoSensitest-2G media and CLSI M27A-3. Microdilution plates were read after 24 and 48 h. The following test parameters were evaluated: fks hot spot mutants overlapping the wild-type distribution, distance between the two populations, number of very major errors (VMEs; fks mutants misclassified as susceptible), and major errors (MEs; wild-type isolates classified as resistant) using a wild-type-upper-limit value (WT-UL) (two twofold-dilutions higher than the MIC(50)) as the susceptibility breakpoint. The methods with the lowest number of errors (given as VMEs/MEs) across the three echinocandins were CLSI (12%/1%), agar dilution with RPMI-2G medium (14%/0%), and Etest with RPMI-2G medium (8%/3%). The fewest errors overall were observed for anidulafungin (4%/1% for EUCAST, 4%/3% for CLSI, and 3%/9% for Etest with RPMI-2G). For micafungin, VME rates of 10 to 71% were observed. For caspofungin, agar dilution with either medium was superior (VMEs/MEs of 0%/1%), while CLSI, EUCAST with IsoSensitest-2G medium, and Etest were less optimal (VMEs of 7%, 10%, and 10%, respectively). Applying the CLSI breakpoint (S

In Vitro Susceptibility Testing in Fungi: a Global Perspective on a Variety of Methods

Mycoses. Jan, 2010  |  Pubmed ID: 20078786

Candida and Aspergillus species are the most common causes of invasive fungal infections in immunocompromised patients. The introduction of new antifungal agents and recent reports of resistance emerging during treatment have highlighted the need for in vitro susceptibility testing. For some drugs, there is a supporting in vitro-in vivo correlation available from studies of clinical efficacy. Both intrinsic and emergent antifungal drug resistance are encountered. Various testing procedures have been proposed, including macrodilution and microdilution, agar diffusion, disk diffusion and Etest. Early recognition of infections caused by pathogens that are resistant to one or more antifungals is highly warranted to optimise treatment and patient outcome.

Invasive Fungal Infections in Pediatric Patients: a Review Focusing on Antifungal Therapy

Expert Review of Anti-infective Therapy. Feb, 2010  |  Pubmed ID: 20109043

Invasive fungal infections in children appear to have increased over the past few decades. Substantial differences compared with adult patients in risk factors, pathogen epidemiology, pathophysiology and, most notably, pharmacokinetics of antifungal agents require age-adapted management and treatment approaches. The present article reviews these aspects for invasive aspergillosis and invasive candidiasis, the two most common life-threatening fungal infections.

Secretion of a Fungal Protease Represents a Complement Evasion Mechanism in Cerebral Aspergillosis

Molecular Immunology. Apr, 2010  |  Pubmed ID: 20303595

Complement represents a central immune weapon in the brain, but the high lethality of cerebral aspergillosis indicates a low efficacy of the antifungal complement attack. Studies with cerebrospinal fluid (CSF) samples derived from a patient with cerebral aspergillosis showed a degradation of complement proteins, implying that Aspergillus might produce proteases to evade their antimicrobial potency. Further investigations of this hypothesis showed that Aspergillus, when cultured in CSF to simulate growth conditions in the brain, secreted a protease that can cleave various complement proteins. Aspergillus fumigatus, the most frequent cause of cerebral aspergillosis, destroyed complement activity more efficiently than other Aspergillus species. The degradation of complement in CSF resulted in a drastic reduction of the capacity to opsonize fungal hyphae. Furthermore, the Aspergillus-derived protease could diminish the amount of complement receptor CR3, a surface molecule to mediate eradication of opsonized pathogens, on granulocytes and microglia. The lack of these prerequisites caused a significant decrease in phagocytosis of primary microglia. Additional studies implied that the complement-degrading activity shares many characteristics with the previously described alkaline protease Alp1. To improve the current therapy for cerebral aspergillosis, we tried to regain the antifungal effects of complement by repressing the secretion of this degrading activity. Supplementation of CSF with nitrogen sources rescued the complement proteins and abolished any cleavage. Glutamine or arginine are of special interest for this purpose since they represent endogenous substances in the CNS and might be included in a future supportive therapy to reduce the high lethality of cerebral aspergillosis.

68Ga-siderophores for PET Imaging of Invasive Pulmonary Aspergillosis: Proof of Principle

Journal of Nuclear Medicine : Official Publication, Society of Nuclear Medicine. Apr, 2010  |  Pubmed ID: 20351354

The diagnosis of invasive pulmonary aspergillosis (IPA) is difficult and lacks specificity and sensitivity. In the pathophysiology of Aspergillus fumigatus, iron plays an essential role as a nutrient during infection. A. fumigatus uses a specific and highly efficient iron uptake mechanism based on iron-complexing ferric ion Fe(III) siderophores, which are a requirement for A. fumigatus virulence. We aimed to evaluate the potential of siderophores radiolabeled with (68)Ga, a positron emitter with complexing properties comparable to those of Fe(III), as a radiopharmaceutical for imaging IPA.

EUCAST Breakpoints for Antifungals

Drug News & Perspectives. Mar, 2010  |  Pubmed ID: 20369073

Susceptibility testing of fungi and development of interpretative breakpoints has become increasingly important due to the growing incidence of invasive fungal infections, the number and classes of antifungals, and the emerging reports of acquired resistance. The subcommittee on antifungal susceptibility testing of the European Committee on Antibiotic Susceptibility Testing (EUCAST) has developed standards for susceptibility testing of fermentative yeasts and molds as well as proposing breakpoints for fluconazole and voriconazole against Candida. The aim of this work is to describe the EUCAST process of setting breakpoints for antifungals. Five aspects are evaluated during the process of developing breakpoints: 1) the most common dosage used in each European country, 2) the definition of the wild-type population for each target microorganism at the species level and the determination of epidemiological cutoffs, 3) the drug's pharmacokinetics and 4) pharmacodynamics, including Monte Carlo simulations, and 5) the correlation of MICs with clinical outcome of patients treated with the compound. When insufficient data are available (e.g., due to lack of information on the clinical outcome of infections caused by isolates with an elevated MIC), epidemiological cutoff values, rather than breakpoints, are recommended until the necessary information becomes available.

Potential Synergistic Activity of Antimycotic Substances in Combination with Human Platelets Against Aspergillus Fumigatus

The Journal of Antimicrobial Chemotherapy. Jun, 2010  |  Pubmed ID: 20378674

Candida Albicans and Its Metabolite Gliotoxin Inhibit Platelet Function Via Interaction with Thiols

Thrombosis and Haemostasis. Aug, 2010  |  Pubmed ID: 20431851

Platelets bind to Candida albicans, the major cause of candidiasis. But in contrast to other microorganisms the fungus does not aggregate platelets. Gliotoxin (GT), which possesses immunosuppressive properties, is produced by various fungi, including the opportunistic pathogens Aspergillus fumigatus and C. albicans . Its mode of action involves the formation of mixed disulfides with host proteins. Disulfide exchanges play an important role in platelet activation. Therefore, the effect of C. albicans and GT on platelet function was tested. C. albicans yeast cells (5,000-10,000 cells/microl) and GT, in pathophysiologically relevant concentrations (0.05-0.5 microM), inhibited platelet fibrinogen binding, anti gp IIb/IIIa antibody PAC-1 binding, aggregation and procoagulant activity in a dose-dependent manner. Alpha granule release, measured via CD62P surface expression, was not affected. Addition of reduced glutathione partially counteracted the effect of C. albicans and GT on platelet fibrinogen binding and platelet aggregation. The C. albicans metabolite GT features antithrombotic properties in addition to its immunosuppressive functions. Since treatment with reduced glutathione partially counteracted the inhibitory effect of C. albicans yeast cells and GT on platelet fibrinogen binding, the antithrombotic activity is likely to depend on the disulfide bridge of this mycotoxin. GT production by C. albicans could contribute to its survival in the blood stream during vascular infections. The knowledge of the underlying mechanisms of the antithrombotic properties might help to treat fungal infections as well as thrombosis.

In Vitro Susceptibility Testing in Aspergillus Species: an Update

Future Microbiology. May, 2010  |  Pubmed ID: 20441550

Aspergillus species are the most common causes of invasive mold infections in immunocompromised patients. The introduction of new antifungal agents and recent reports of resistance emerging during treatment of Aspergillus infections have highlighted the need for in vitro susceptibility testing. Various testing procedures have been proposed, including macro- and micro-dilution, disk diffusion, Etest (AB Biodisk, Sweden) and other commercial tests. Although Aspergillus species are generally susceptible to various compounds, intrinsic and acquired resistance has been documented. Amphotericin B has limited activity against Aspergillus terreus and Aspergillus nidulans. Not surprisingly, continued use of azole-based drugs has the undesirable consequence of elevating the resistance of subsequent isolates from these patients. Several species in the Aspergillus fumigatus complex appear to be resistant to azoles; there is evidence of in vitro and in vivo correlation. Each in vitro susceptibility testing method has its own advantages and disadvantages. Etest is easy to perform and use on a daily basis, yet it is expensive. Disk diffusion is the most attractive alternative method to date, yet we lack sufficient data for aspergilli. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical Laboratory Standard Institute (CLSI) have produced reproducible reference testing methods. This article reviews the available methods for antifungal susceptibility testing in Aspergillus spp. as well as the scant data regarding the clinical implications of in vitro testing.

Comparison of Caspofungin MICs by Means of EUCAST Method EDef 7.1 Using Two Different Concentrations of Glucose

Antimicrobial Agents and Chemotherapy. Jul, 2010  |  Pubmed ID: 20479199

According to the product insert for Cancidas (caspofungin acetate), the drug must not be diluted in solutions containing glucose as this decreases caspofungin stability. The aim of this study was to compare caspofungin MICs for a collection of yeasts by means of EUCAST method EDef7.1 but using two different concentrations of glucose: 2% versus 0.2%. MICs were identical or within one 2-fold dilution for 93 out of 95 strains (97.9%), showing that glucose does not interfere with susceptibility.

Environmental Study of Azole-resistant Aspergillus Fumigatus and Other Aspergilli in Austria, Denmark, and Spain

Antimicrobial Agents and Chemotherapy. Nov, 2010  |  Pubmed ID: 20805399

A single mechanism of azole resistance was shown to predominate in clinical and environmental Aspergillus fumigatus isolates from the Netherlands, and a link to the use of azoles in the environment was suggested. To explore the prevalence of azole-resistant A. fumigatus and other aspergilli in the environment in other European countries, we collected samples from the surroundings of hospitals in Copenhagen, Innsbruck, and Madrid, flowerbeds in an amusement park in Copenhagen, and compost bags purchased in Austria, Denmark, and Spain and screened for azole resistance using multidish agars with itraconazole, voriconazole, and posaconazole. EUCAST method E.DEF 9.1 was used to confirm azole resistance. The promoter and entire coding sequence of the cyp51A gene were sequenced to identify azole-resistant A. fumigatus isolates. A. fumigatus was recovered in 144 out of 185 samples (77.8%). Four A. fumigatus isolates from four Danish soil samples displayed elevated azole MICs (8%), and all harbored the same TR/L98H mutation of cyp51A. One A. lentulus isolate with voriconazole MIC of 4 mg/liter was detected in Spain. No azole-resistant aspergilli were detected in compost. Finally, A. terreus was present in seven samples from Austria. Multi-azole-resistant A. fumigatus is present in the environment in Denmark. The resistance mechanism is identical to that of environmental isolates in the Netherlands. No link to commercial compost could be detected. In Spain and Austria, only Aspergillus species with intrinsic resistance to either azoles or amphotericin B were found.

In Vitro Susceptibility Testing in Fungi: What is Its Role in Clinical Practice?

Current Infectious Disease Reports. Nov, 2010  |  Pubmed ID: 21308547

An increasing number of patients are undergoing transplantation procedures or receiving aggressive immunosuppression and chemotherapy. The growing population of immunocompromised hosts has led to a rise in the prevalence of invasive fungal infections due to yeasts and molds. The introduction of new antifungal agents and recent reports of resistance emerging during treatment of fungal infections have highlighted the need for in vitro susceptibility testing. Various testing procedures have been proposed, including macrodilution and microdilution, agar diffusion, disk diffusion, and Etest (AB Biodisk, Solna, Sweden). Establishing clinical correlation with antifungal susceptibility testing, however, is a huge challenge because susceptibility techniques do not take into account the dynamic and complex biology of fungi exposed to an antifungal in vivo. This paper reviews the available methods for antifungal susceptibility testing of yeasts and filamentous fungi and the data regarding the clinical implications of in vitro testing.

Platelets Enhance Activity of Antimycotic Substances Against Non-Aspergillus Fumigatus Aspergillus Species in Vitro

Medical Mycology. Feb, 2011  |  Pubmed ID: 20795763

Platelets are known to be part of haemostasis but they are also players in innate host defense. Recently, we observed that platelets attenuate the virulence of Aspergillus spp. in vitro. However, little is known about the antifungal effects of platelets in the presence of antimycotics against non-A. fumigatus Aspergillus species. We therefore investigated whether platelets increase the in vitro activity of amphotericin B, voriconazole, posaconazole and caspofungin against two clinical isolates each of Aspergillus flavus, Aspergillus terreus and Aspergillus niger. The antifungal activity was evaluated by assessing germination percentages, hyphal elongation and hyphal damage by use of XTT. The combination of platelets plus amphotericin B significantly (P < 0.05) enhanced the reduction of germination percentage compared to either substance alone. Among triazoles, voriconazole exhibited significant effects with platelets for all tested aspergilli. Overall, these findings suggest that among the tested antimycotic substances, amphotericin B in combination with platelets has enhancing effects in reducing germination and hyphal elongation in the tested non-A. fumigatus Aspergillus species. These data indicate that platelets act beneficially with antimycotics in an early stage of fungal growth by blocking and/or delaying fungal germination and hyphal elongation; both crucial mechanisms in the development of invasive fungal disease.

Detection and Investigation of Invasive Mould Disease

The Journal of Antimicrobial Chemotherapy. Jan, 2011  |  Pubmed ID: 21177400

A comprehensive review of diagnostic techniques for opportunistic systemic mycoses focused on invasive mould disease in immunocompromised patients is presented. We first analysed conventional diagnostic methods, such as microscopy examination, culture and radiology, underlining their limitations, which have led to the development of alternative methods, such as the detection of fungal components. Among these we highlight fungal antigen and DNA quantification, which make it possible to detect infections early and start appropriate treatment. We also briefly review the methods for carrying out susceptibility tests for antifungal drugs, including reference procedures, commercial techniques and their indications. Furthermore, we analyse the recommendations for therapeutic drug monitoring of antifungal agents in body fluids.

Multicenter, Prospective Clinical Evaluation of Respiratory Samples from Subjects at Risk for Pneumocystis Jirovecii Infection by Use of a Commercial Real-time PCR Assay

Journal of Clinical Microbiology. May, 2011  |  Pubmed ID: 21367988

Pneumocystis jirovecii pneumonia (PCP) is a common opportunistic infection. Microscopic diagnosis, including diagnosis using the Merifluor-Pneumocystis direct fluorescent antigen (MP-DFA) test, has limitations. Real-time PCR may assist in diagnosis, but no commercially validated real-time PCR assay has been available to date. MycAssay Pneumocystis is a commercial assay that targets the P. jirovecii mitochondrial large subunit (analytical detection limit, ≤ 3.5 copies/μl of sample). A multicenter trial recruited 110 subjects: 54 with transplants (40 with lung transplants), 32 with nonmalignant conditions, 13 with leukemia, and 11 with solid tumors; 9 were HIV positive. A total of 110 respiratory samples (92% of which were bronchoalveolar lavage [BAL] specimens) were analyzed by PCR. Performance was characterized relative to investigator-determined clinical diagnosis of PCP (including local diagnostic tests), and PCR results were compared with MP-DFA test results for 83 subjects. Thirteen of 14 subjects with PCP and 9/96 without PCP (including 5 undergoing BAL surveillance after lung transplantation) had positive PCR results; sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) were 93%, 91%, 59%, and 99%, respectively. Fourteen of 83 subjects for whom PCR and MP-DFA test results were available had PCP; PCR sensitivity, specificity, PPV, and NPV were 93%, 90%, 65%, and 98%, respectively, and MP-DFA test sensitivity, specificity, PPV, and NPV were 93%, 100%, 100%, and 98%. Of the 9 PCR-positive subjects without PCP, 1 later developed PCP. The PCR diagnostic assay compares well with clinical diagnosis using nonmolecular methods. Additional positive results compared with the MP-DFA test may reflect low-level infection or colonization.

High-frequency Triazole Resistance Found In Nonculturable Aspergillus Fumigatus from Lungs of Patients with Chronic Fungal Disease

Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America. May, 2011  |  Pubmed ID: 21467016

Oral triazole therapy is well established for the treatment of invasive (IPA), allergic (ABPA), and chronic pulmonary (CPA) aspergillosis, and is often long-term. Triazole resistance rates are rising internationally. Microbiological diagnosis of aspergillosis is limited by poor culture yield, leading to uncertainty about the frequency of triazole resistance.

Epidemiology and Antifungal Resistance in Invasive Aspergillosis According to Primary Disease: Review of the Literature

European Journal of Medical Research. Apr, 2011  |  Pubmed ID: 21486729

Aspergilli, less susceptible to antifungals emerge and resistance to azoles have been found mainly in Aspergillus fumigatus; this has launched a new phase in handling aspergillosis. Resistant strains have currently been reported from Belgium, Canada, China, Denmark, France, Norway, Spain, Sweden, The Netherlands, UK and the USA. Centres in the UK (Manchester) and The Netherlands (Nijmegen) have described particularly high frequencies (15 and 10% respectively), and a significant increase in azole resistance in recent years. The reason of this high incidence may be due to long term azole therapy in patients with chronic aspergillosis in Manchester, and due to high use of agricultural azoles in Nijmegen. The primary underlying mechanism of resistance is as a result of alterations in the cyp51A target gene, with a variety of mutations found in clinical isolates and one genotype identified in the environmental (LH98). Reports on well documented in vitro and in vivo resistance to echinocandins are rare for Aspergillus species and resistance may be under-diagnosed as susceptibility testing is less frequently performed due to technical reasons.

Susceptibility of Candida Species and Various Moulds to Antimycotic Drugs: Use of Epidemiological Cutoff Values According to EUCAST and CLSI in an 8-year Survey

Medical Mycology. Nov, 2011  |  Pubmed ID: 21619497

A collection of 2,834 isolates of Candida spp. and 1,079 isolates of Aspergillus spp. and other moulds that were recovered between 2000 and 2007 in Tyrol, Austria, were examined for their susceptibility to antifungal drugs. The susceptibility of Candida spp. to amphotericin B (AMB), caspofungin (CPF), fluconazole (FLC), and voriconazole (VRC) were studied, while filamentous fungi were tested against AMB, CPF, VRC, itraconazole (ITC), and posaconazole (POS). As EUCAST and CLSI are currently revising their breakpoints for several antifungal agents, epidemiological cutoff values (ECVs) of these two guidelines were used to examine trends in susceptibility. For Candida spp. we noted increases in the percentage of non-wild type isolates which were resistant to CPF, FLC, and VRC. Most noticeably, we observed a change in: C. tropicalis (from 0.9-3.8%) and C. parapsilosis (from 4.0-6.0%) relative to CPF; C. parapsilosis (from 0.8-3.4%) and C. glabrata (from 11.0-20%) against FLC; and C. glabrata (from 3.0-12.0%) for VRC. Among the moulds, most Aspergillus spp. isolates were found to be susceptible to VRC, ITC, and POS, while AMB and POS were confirmed to be the most effective agents against zygomycetes. EUCAST and CLSI should continue their efforts to harmonize their methods of antimicrobial susceptibility testing (AST) and to define additional and shareable epidemiological cutoff values and clinical breakpoints.

Epidemiology of Invasive Fungal Infections in the Mediterranean Area

Mediterranean Journal of Hematology and Infectious Diseases. 2011  |  Pubmed ID: 21625305

Although Candida species remain the relevant cause of IFI, other fungi (especially moulds) have become increasingly prevalent. In particular, Aspergillus species are the leading cause of mould infections but also Glomeromycota (formerly Zygomycetes) and Fusarium species are increasing in frequency, and are associated with high mortality rates. Many of these emerging infections occur as breakthrough infections in patients treated with new antifungal drugs. The causative pathogens, incidence rate and severity are dependent on the underlying condition, as well as on the geographic location of the patient population. France and Italy show the highest incident rates of Fusarium infections in Europe, following the US, where numbers are still increasing. Scedosporium prolificans, which primarily is found in soil in Spain and Australia, is most frequently isolated from blood cultures in a Spanish hospital. Geotrichum capitatum represents another species predominantly found in Europe with especially high rates in Mediterranean countries. The increasing resistance to antifungal drugs especially of these new emerging pathogens is a severe problem for managing these IFIs.

Diagnosis and Therapy of Candida Infections: Joint Recommendations of the German Speaking Mycological Society and the Paul-Ehrlich-Society for Chemotherapy

Mycoses. Jul, 2011  |  Pubmed ID: 21672038

Invasive Candida infections are important causes of morbidity and mortality in immunocompromised and hospitalised patients. This article provides the joint recommendations of the German-speaking Mycological Society (Deutschsprachige Mykologische Gesellschaft, DMyKG) and the Paul-Ehrlich-Society for Chemotherapy (PEG) for diagnosis and treatment of invasive and superficial Candida infections. The recommendations are based on published results of clinical trials, case-series and expert opinion using the evidence criteria set forth by the Infectious Diseases Society of America (IDSA). Key recommendations are summarised here: The cornerstone of diagnosis remains the detection of the organism by culture with identification of the isolate at the species level; in vitro susceptibility testing is mandatory for invasive isolates. Options for initial therapy of candidaemia and other invasive Candida infections in non-granulocytopenic patients include fluconazole or one of the three approved echinocandin compounds; liposomal amphotericin B and voriconazole are secondary alternatives because of their less favourable pharmacological properties. In granulocytopenic patients, an echinocandin or liposomal amphotericin B is recommended as initial therapy based on the fungicidal mode of action. Indwelling central venous catheters serve as a main source of infection independent of the pathogenesis of candidaemia in the individual patients and should be removed whenever feasible. Pre-existing immunosuppressive treatment, particularly by glucocorticosteroids, ought to be discontinued, if feasible, or reduced. The duration of treatment for uncomplicated candidaemia is 14 days following the first negative blood culture and resolution of all associated symptoms and findings. Ophthalmoscopy is recommended prior to the discontinuation of antifungal chemotherapy to rule out endophthalmitis or chorioretinitis. Beyond these key recommendations, this article provides detailed recommendations for specific disease entities, for antifungal treatment in paediatric patients as well as a comprehensive discussion of epidemiology, clinical presentation and emerging diagnostic options of invasive and superficial Candida infections.

Gliotoxin As Putative Virulence Factor and Immunotherapeutic Target in a Cell Culture Model of Cerebral Aspergillosis

Molecular Immunology. Sep, 2011  |  Pubmed ID: 21803423

The mycotoxin gliotoxin is an important metabolite produced by Aspergillus fumigatus, but its precise role in the pathogenesis of cerebral aspergillosis is not yet determined. We could demonstrate that growth in cerebrospinal fluid (CSF) induced the production and secretion of significant amounts of gliotoxin by A. fumigatus. These concentrations of 590-720nM were sufficient to reduce the viability of astrocytes and neurons, as well as of primary microglia, already after few hours of incubation. Annexin staining and electron microscopy revealed the induction of apoptosis rather than necrosis as the relevant mode of gliotoxin action in the brain. Furthermore, even a low gliotoxin concentration of 100nM, which was subtoxic for astrocytes, was able to significantly down-modulate the phagocytic capacity of astrocytes. In order to improve the current antimycotic therapy of cerebral aspergillosis by supporting innate immunity in the fight against Aspergillus, we aimed to neutralize the toxic potency of gliotoxin towards different brain cell types. Compounds such as dithiothreitol (DTT) or glutathione that reduce the internal disulfide bond of gliotoxin were shown here to be able to interfere with the gliotoxin-induced decrease of cell viability and to save the cells from induction of apoptosis. Thus, exploration of these substances may lead to novel approaches for adjunctive treatment of cerebral aspergillosis.

Candida Albicans Hgt1p, a Multifunctional Evasion Molecule: Complement Inhibitor, CR3 Analogue, and Human Immunodeficiency Virus-binding Molecule

The Journal of Infectious Diseases. Sep, 2011  |  Pubmed ID: 21844307

The complement system is tightly controlled by several regulators. Two of these, factor H (FH) and C4b-binding protein (C4BP), can be acquired by pathogens conveying resistance to complement attack. The aim of the study was to characterize the FH binding molecule of Candida albicans, a potentially life-threatening yeast.

Biopsy Procedures for Molecular Tissue Diagnosis of Invasive Fungal Infections

Current Infectious Disease Reports. Dec, 2011  |  Pubmed ID: 21948145

The incidence of invasive fungal infections (IFI) has increased substantially and the epidemiology has changed dramatically in recent years. Candida albicans is still most important, but non-albicans species, Aspergillus species, Glomeromycota (formerly Zygomycetes) and Fusarium species are an increasing cause of IFIs. Due to this growing diversity, the identification of the causative organism to genus and species level is important to perform best and adequate treatment. The early, sensitive and specific detection of IFIs remains challenging and current conventional methods are limited. The golden standard for the definite diagnosis of proven pulmonary infection remains either histopathologic, cytopathologic or direct tissue examination. Invasive procedures are necessary to obtain reliable specimens and biopsies may be taken percutaneously, bronchoscopically, via open surgery or via video-assisted thorascopic surgery. Molecular methods, like PCR or in situ hybridization, are a promising diagnostic tool for rapid and reliable species identification and should be performed in addition to microscopic examination and culture to increase the sensitivity for the diagnosis of IFI. Combining culture, microscopy, serology, and PCR in lung tissues and/or bronchial samples will increase the diagnostic yield by 99%. Here, we give an overview of biopsy procedures for molecular tissue diagnosis of IFI.

Triazole Antifungal Agents in Invasive Fungal Infections: a Comparative Review

Drugs. Dec, 2011  |  Pubmed ID: 22141384

Invasive fungal disease continues to be a problem associated with significant morbidity and high mortality in immunocompromised and, to a lesser extent, immunocompetent individuals. Triazole antifungals have emerged as front-line drugs for the treatment and prophylaxis of many systemic mycoses. Fluconazole plays an excellent role in prophylaxis, empirical therapy, and the treatment of both superficial and invasive yeast fungal infections. Voriconazole is strongly recommended for pulmonary invasive aspergillosis. Posaconazole shows a very wide spectrum of activity and its primary clinical indications are as salvage therapy for patients with invasive aspergillosis and prophylaxis for patients with neutropenia and haematopoietic stem-cell transplant recipients. Itraconazole also has a role in the treatment of fungal skin and nail infections as well as dematiaceous fungi and endemic mycoses. Fluconazole and voriconazole are well absorbed and exhibit high oral bioavailability, whereas the oral bioavailability of itraconazole and posaconazole is lower and more variable. Posaconazole absorption depends on administration with a high-fat meal or nutritional supplements. Itraconazole and voriconazole undergo extensive hepatic metabolism involving the cytochrome P450 system. The therapeutic window for triazoles is narrow, and inattention to their pharmacokinetic properties can lead to drug levels too low for efficacy or too high for good tolerability or safety. This makes these agents prime candidates for therapeutic drug monitoring (TDM). Target drug concentrations for voriconazole and itraconazole should be >1 μg/mL and for posaconazole >1.5 μg/mL for treatment. Blood should be drawn once the patient reaches steady state, which occurs after 5 and 7 days of triazole therapy. Routine TDM of fluconazole is not required given its highly favourable pharmacokinetic profile and wide therapeutic index. The aim of this review is to provide a brief update on the pharmacology, activity, clinical efficacy, safety and cost of triazole agents (itraconazole, fluconazole, voriconazole and posaconazole) and highlight the clinical implications of similarities and differences.

Caspofungin: when and How? The Microbiologist's View

Mycoses. Jan, 2012  |  Pubmed ID: 21668518

The echinocandins are antifungal agents, which act by inhibiting the synthesis of β-(1,3)-D-glucan, an integral component of fungal cell walls. Caspofungin, the first approved echinocandin, demonstrates good in vitro and in vivo activity against a range of Candida species and is an alternative therapy for Aspergillus infections. Caspofungin provides an excellent safety profile and is therefore favoured in patients with moderately severe to severe illness, recent azole exposure and in those who are at high risk of infections due to Candida glabrata or Candida krusei. In vivo/in vitro resistance to caspofungin and breakthrough infections in patients receiving this agent have been reported for Candida and Aspergillus species. The types of pathogens and the frequency causing breakthrough mycoses are not well delineated. Caspofungin resistance resulting in clinical failure has been linked to mutations in the Fksp subunit of glucan synthase complex. European Committee for Antimicrobial Susceptibility Testing and Clinical and Laboratory Standards Institute need to improve the in vitro susceptibility testing methods to detect fks hot spot mutants. Caspofungin represents a significant advance in the care of patients with serious fungal infections.

Airborne Fungus Exposure Prior to Hospitalisation As Risk Factor for Mould Infections in Immunocompromised Patients

Mycoses. May, 2012  |  Pubmed ID: 21797937

The aim of this study was to investigate the relationship between fungal exposure prior to hospitalisation and ensuing onset of invasive mould infections (IMI) in patients at risk. Patients admitted to the Department of Haematology, Oncology and Transplant Surgery of the Medical University Innsbruck received a questionnaire regarding fungal exposure prior to hospital stay. Questions inquired heavy fungal exposures up to 5 days before hospitalisation. A total of 234 patients were enrolled in this study. Multiple fungus exposures were associated with the onset of community-acquired IMI in patients with haematological malignancies. In univariate analysis, haematological malignancies (P = 0.013) and allergy to dust, pollen or moulds (P = 0.015) were significantly associated with fungal infections. In multivariate analysis, logistic regression showed that haematological patients (P = 0.015) and patients with allergy (P = 0.015) were significantly more frequently infected with fungi. Hospital-independent fungal sources highlight risk-factors for IMI in severe immunocompromised patients and the rate of community-acquired IMI does increase.

Inhibition of Human Immunodeficiency Virus Replication by Cell Membrane-crossing Oligomers

Molecular Medicine (Cambridge, Mass.). 2012  |  Pubmed ID: 22105607

Although rapidly becoming a valuable tool for gene silencing, regulation or editing in vitro, the direct transfer of small interfering ribonucleic acids (siRNAs) into cells is still an unsolved problem for in vivo applications. For the first time, we show that specific modifications of antisense oligomers allow autonomous passage into cell lines and primary cells without further adjuvant or coupling to a cell-penetrating peptide. For this reason, we termed the specifically modified oligonucleotides "cell membrane-crossing oligomers" (CMCOs). CMCOs targeted to various conserved regions of human immunodeficiency virus (HIV)-1 were tested and compared with nontargeting CMCOs. Analyses of uninfected and infected cells incubated with labeled CMCOs revealed that the compounds were enriched in infected cells and some of the tested CMCOs exhibited a potent antiviral effect. Finally, the CMCOs did not exert any cytotoxicity and did not inhibit proliferation of the cells. In vitro, our CMCOs are promising candidates as biologically active anti-HIV reagents for future in vivo applications.

Interlaboratory Comparison of PCR-based Identification of Candida and Aspergillus DNA in Spiked Blood Samples

Mycoses. Sep, 2012  |  Pubmed ID: 22248125

Despite PCR per se being a powerful and sensitive technique, regarding the detection of fungi in patients' blood, no consensus for a standardised PCR protocol yet exists. To complement other ongoing or accomplished studies which tackle this problem, the German Reference Center for Systemic Mycoses conducted an interlaboratory comparison starting with blood samples spiked with fungal cell elements. Altogether, six laboratories using in-house PCR-protocols from Germany and Austria participated in the trial. Blood samples were spiked with vital cells of Candida albicans or Aspergillus fumigatus. Candida was used in the yeast form, whereas Aspergillus cells were either spiked as conidia or as very young germlings, also known as smoo cells. Spiked blood samples contained between 10 and 10 000 cells ml(-1). Depending on the techniques used for fungal cell disruption and DNA-amplification, detection quality was variable between laboratories, but also differed within single laboratories in different trials particularly for samples spiked with less than 100 cells ml(-1). Altogether, at least regarding the detection of A. fumigatus, two of six laboratories showed constant reliable test results also with low fungal cell number spiked samples. Protocols used by these labs do not differ substantially from others. However, as particularities, one protocol included a conventional phenol chloroform extraction during the DNA preparation process and the other included a real time PCR-protocol based on FRET probes. Other laboratory comparisons on the basis of clinical samples should follow to further evaluate the procedures. The difficulties and problems of such trials in general are discussed.

Aspergillus Terreus: How Inoculum Size and Host Characteristics Affect Its Virulence

The Journal of Infectious Diseases. Apr, 2012  |  Pubmed ID: 22438395

[Diagnosis of Aspergillus Infections in Hematology and Oncology]

Mycoses. Jul, 2012  |  Pubmed ID: 22519626

Preclinical Evaluation of Two 68Ga-siderophores As Potential Radiopharmaceuticals for Aspergillus Fumigatus Infection Imaging

European Journal of Nuclear Medicine and Molecular Imaging. Jul, 2012  |  Pubmed ID: 22526953

Invasive pulmonary aspergillosis is mainly caused by Aspergillus fumigatus, and is one of the major causes of morbidity and mortality in immunocompromised patients. The mortality associated with invasive pulmonary aspergillosis remains high, mainly due to the difficulties and limitations in diagnosis. We have shown that siderophores can be labelled with (68)Ga and can be used for PET imaging of A. fumigatus infection in rats. Here we report on the further evaluation of the most promising (68)Ga-siderophore candidates, triacetylfusarinine (TAFC) and ferrioxamine E (FOXE).

Rapid Detection of Bloodstream Pathogens by Real-time PCR in Patients with Sepsis

Wiener Klinische Wochenschrift. Apr, 2012  |  Pubmed ID: 22527822

Rapid detection of bloodstream infections is an important issue for a better patient outcome. The aim of our study was thus to evaluate the LightCycler SeptiFast assay for diagnosis of bloodstream pathogens in a tertiary hospital in Western Austria. The 71 blood samples of 61 patients with presumed sepsis were investigated and compared with conventional blood culture system results. In both assays, 51 samples (71.8 %) were negative. In 20 positive samples (28.2 %), 10 different pathogens were detected by either blood culture system or SeptiFast assay or by both methods. Five samples were positive in both assays. The agreement rate of blood culture system and SeptiFast assay was 78.9 %, the negative predictive value of SeptiFast assay versus blood culture system was 0.94, sensitivity was 0.63, and specificity 0.81. In 12 samples where a positive SeptiFast assay and a negative blood culture system result were obtained, the same pathogens as identified by SeptiFast assay were detected in samples from other body sites suggesting a correct positive detection. In 11.3 % of cases, the SeptiFast assay resulted in an adjustment of the patients' therapy. In 3 samples, the blood culture assay was positive whereas the SeptiFast assay yielded negative results. In two of these cases, the pathogens involved were not included in the SeptiFast detection list, in the third case SeptiFast assay failed to detect Candida glabrata.Thus we recommend the SeptiFast assay as a valuable tool for rapid diagnosis of bloodstream infections in addition to, but not as replacement for, the blood culture test.

Characterization of the Cellular Immune Responses to Rhizopus Oryzae with Potential Impact on Immunotherapeutic Strategies in Hematopoietic Stem Cell Transplantation

The Journal of Infectious Diseases. Jul, 2012  |  Pubmed ID: 22529315

Infections due to mucormycetes have a poor outcome, in particular in allogeneic hematopoietic stem cell transplantation (HSCT). In order to evaluate the cellular host response against mucormycetes, we enriched and cultivated anti-Rhizopus oryzae T cells from healthy individuals. These cells were characterized as memory/effector T(H)1 cells, they proliferated upon restimulation, they exhibited cross-reactivity to some but not all Mucorales species tested, and they increased the activity of phagocytes. Compared with the original cell fraction, the generated cells exhibited significant lower alloreactivity. Our data may form the basis for further investigations, which may ultimately lead to adoptive immunotherapeutic strategies for allogeneic HSCT recipients suffering from mucormycosis.

Comparison of Dimethyl Sulfoxide and Water As Solvents for Echinocandin Susceptibility Testing by the EUCAST Methodology

Journal of Clinical Microbiology. Jul, 2012  |  Pubmed ID: 22535988

Ninety-six strains of Candida, including 29 resistant and 67 susceptible isolates with mutations in the FKS1 and FKS2 genes were tested by the European Committee on Antibiotic Susceptibility Testing EDef 7.1 and 7.2 methodologies to determine the impact on the MIC when water was replaced with dimethyl sulfoxide (DMSO) as the solvent for caspofungin and micafungin. The MICs were significantly lower and the MIC ranges were narrower when DMSO was used as the solvent. The use of DMSO may help to better discriminate between susceptible and resistant populations.

Microbial Challenge Tests on Nonradioactive TiO2-based 68Ge/68Ga Generator Columns

Nuclear Medicine Communications. Aug, 2012  |  Pubmed ID: 22546876

The aim of this study was to test the survival of microorganisms in eluents used for (68)Ge/(68)Ga generators and for regeneration of nonradioactive (68)Ge/(68)Ga generator columns after high microbial load. Nonradioactive generator columns were loaded with various microorganisms and tested to determine whether the microorganisms were proliferating or surviving in eluates of the columns.

EUCAST Technical Note on the EUCAST Definitive Document EDef 7.2: Method for the Determination of Broth Dilution Minimum Inhibitory Concentrations of Antifungal Agents for Yeasts EDef 7.2 (EUCAST-AFST)

Clinical Microbiology and Infection : the Official Publication of the European Society of Clinical Microbiology and Infectious Diseases. Jul, 2012  |  Pubmed ID: 22563750

The European Committee on Antimicrobial Susceptibility Testing-Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST) has revised the EDef 7.1 document on the method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for fermentative yeasts. Changes are: dimethylsulphoxide is now the recommended solvent for caspofungin, micafungin and fluconazole; the shelf-life of plates containing the echinocandins prepared from stock solutions in dimethylsulphoxide is extended to 6 months at -80°C; testing of amphotericin and Cryptococcus has been incorporated; and minimum inhibitory concentration ranges for quality control strains and anidulafungin are included.

European Organization for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) Host Factors and Invasive Fungal Infections in Patients with Haematological Malignancies

The Journal of Antimicrobial Chemotherapy. Aug, 2012  |  Pubmed ID: 22566591

Fulfilment of host factors defined by the revised European Organization for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria is required for establishing the diagnosis of possible or probable invasive fungal infection (IFI). This case-control study evaluates EORTC/MSG host factors among patients with haematological malignancies.

Pharmacodynamics of Voriconazole in a Dynamic in Vitro Model of Invasive Pulmonary Aspergillosis: Implications for in Vitro Susceptibility Breakpoints

The Journal of Infectious Diseases. Aug, 2012  |  Pubmed ID: 22634880

Voriconazole is a first-line agent for the treatment of invasive pulmonary aspergillosis (IPA). There are increasing reports of Aspergillus fumigatus isolates with reduced susceptibility to voriconazole.

Diagnosing Pulmonary Aspergillosis in Patients with Hematological Malignancies: a Multicenter Prospective Evaluation of an Aspergillus PCR Assay and a Galactomannan ELISA in Bronchoalveolar Lavage Samples

European Journal of Haematology. Aug, 2012  |  Pubmed ID: 22650156

Diagnosing invasive pulmonary aspergillosis (IPA) remains a challenge in patients with hematological malignancies. The clinical significance of testing bronchoalveolar lavage (BAL) samples both with polymerase chain reaction (PCR) and Aspergillus galactomannan ELISA (GM) is unclear, and the BAL cutoff for GM has not been clearly evaluated yet.

Invasive Fungal Breakthrough Infections, Fungal Colonization and Emergence of Resistant Strains in High-risk Patients Receiving Antifungal Prophylaxis with Posaconazole: Real-life Data from a Single-centre Institutional Retrospective Observational Study

The Journal of Antimicrobial Chemotherapy. Sep, 2012  |  Pubmed ID: 22653819

The broad-spectrum triazole posaconazole showed promising results in preventing invasive fungal infections (IFIs) in high-risk patients. Concerns rise over the relevance of breakthrough IFIs (bIFIs) and the emergence of azole-resistant strains. The current retrospective analysis was undertaken to evaluate the incidence of bIFIs and to study fungal colonization and resistance following posaconazole exposure.

In Vivo Emergence of Aspergillus Terreus with Reduced Azole Susceptibility and a Cyp51a M217I Alteration

The Journal of Infectious Diseases. Sep, 2012  |  Pubmed ID: 22782947

Azole resistance in Aspergillus terreus isolates was explored. Twenty related (MB) and 6 unrelated A. terreus isolates were included. CYP51A sequencing and RAPD genotyping was performed. Five MB isolates were itraconazole susceptible, whereas the minimum inhibitory concentrations (MICs) for 15 MB isolates were elevated (1-2 mg/L). Voriconazole and posaconazole MICs were 0.5-4 and 0.06-0.5 mg/L, respectively, for MB isolates but 0.25-0.5 and <0.03-0.06 mg/L, respectively, for controls. Sequencing identified a Cyp51Ap M217I alteration in all 15 isolates with elevated itraconazole MICs. Genotyping showed that 18 of 20 MB isolates were identical and unique, suggesting endogenous origin. In conclusion, itraconazole resistance in A. terreus was linked to an M217I Cyp51A alteration.

Susceptibility Screening of Hyphae-forming Fungi with a New, Easy, and Fast Inoculum Preparation Method

Mycopathologia. Dec, 2012  |  Pubmed ID: 22864604

In vitro susceptibility testing of clinically important fungi becomes more and more essential due to the rising number of fungal infections in patients with impaired immune system. Existing standardized microbroth dilution methods for in vitro testing of molds (CLSI, EUCAST) are not intended for routine testing. These methods are very time-consuming and dependent on sporulating of hyphomycetes. In this multicentre study, a new (independent of sporulation) inoculum preparation method (containing a mixture of vegetative cells, hyphae, and conidia) was evaluated. Minimal inhibitory concentrations (MIC) of amphotericin B, posaconazole, and voriconazole of 180 molds were determined with two different culture media (YST and RPMI 1640) according to the DIN (Deutsches Institut für Normung) microdilution assay. 24 and 48 h MIC of quality control strains, tested per each test run, prepared with the new inoculum method were in the range of DIN. YST and RPMI 1640 media showed similar MIC distributions for all molds tested. MIC readings at 48 versus 24 h yield 1 log(2) higher MIC values and more than 90 % of the MICs read at 24 and 48 h were within ± 2 log(2) dilution. MIC end point reading (log(2 MIC-RPMI 1640)-log(2 MIC-YST)) of both media demonstrated a tendency to slightly lower MICs with RPMI 1640 medium. This study reports the results of a new, time-saving, and easy-to-perform method for inoculum preparation for routine susceptibility testing that can be applied for all types of spore-/non-spore and hyphae-forming fungi.

CHD1 Contributes to Intestinal Resistance Against Infection by P. Aeruginosa in Drosophila Melanogaster

PloS One. 2012  |  Pubmed ID: 22912810

Drosophila SNF2-type ATPase CHD1 catalyzes the assembly and remodeling of nucleosomal arrays in vitro and is involved in H3.3 incorporation in viin vivo during early embryo development. Evidence for a role as transcriptional regulator comes from its colocalization with elongating RNA polymerase II as well as from studies of fly Hsp70 transcription. Here we used microarray analysis to identify target genes of CHD1. We found a fraction of genes that were misregulated in Chd1 mutants to be functionally linked to Drosophila immune and stress response. Infection experiments using different microbial species revealed defects in host defense in Chd1-deficient adults upon oral infection with P. aeruginosa but not upon septic injury, suggesting a so far unrecognized role for CHD1 in intestinal immunity. Further molecular analysis showed that gut-specific transcription of antimicrobial peptide genes was overactivated in the absence of infection in Chd1 mutant flies. Moreover, microbial colonization of the intestine was elevated in Chd1 mutants and oral infection resulted in strong enrichment of bacteria in the body cavity indicating increased microbial passage across intestinal epithelia. However, we did not detect enhanced epithelial damage or alterations of the intestinal stem cell population. Collectively, our data provide evidence that intestinal resistance against infection by P. aeruginosa in Drosophila is linked to maintaining proper balance of gut-microbe interactions and that the chromatin remodeler CHD1 is involved in regulating this aspect.

Antibodies Attenuate the Capacity of Dendritic Cells to Stimulate HIV-specific Cytotoxic T Lymphocytes

The Journal of Allergy and Clinical Immunology. Dec, 2012  |  Pubmed ID: 23063584

Control of HIV is suggested to depend on potent effector functions of the virus-specific CD8(+) T-cell response. Antigen opsonization can modulate the capture of antigen, its presentation, and the priming of specific CD8(+) T-cell responses.

What Paths Are Open for Tackling Increasing Azole Resistance in Aspergillus in the Clinic?

Expert Review of Anti-infective Therapy. Nov, 2012  |  Pubmed ID: 23241178

Interaction of Platelets and Anidulafungin Against Aspergillus Fumigatus

Antimicrobial Agents and Chemotherapy. Jan, 2013  |  Pubmed ID: 23114752

The combination of platelets and anidulafungin at 0.03 μg/ml significantly (P < 0.05) reduced the germination rate and hyphal elongation in Aspergillus fumigatus compared to those with either anidulafungin only or an untreated control. Platelets decreased the expression of the fks gene, which plays an important role in cell wall synthesis. Our results suggest that human platelets plus anidulafungin might contribute to defense against A. fumigatus.

Rhizopus Oryzae Hyphae Are Damaged by Human Natural Killer (NK) Cells, but Suppress NK Cell Mediated Immunity

Immunobiology. Jul, 2013  |  Pubmed ID: 23201314

Mucormycosis has a high mortality and is increasingly diagnosed in hematopoietic stem cell transplant (HSCT) recipients. In this setting, there is a growing interest to restore host defense to combat infections by adoptively transferring donor-derived immunocompetent cells. Natural killer (NK) cells exhibit antitumor and antiinfective activity, but the interaction with Mucormycetes is unknown. Our data demonstrate that both unstimulated and IL-2 prestimulated human NK cells damage Rhizopus oryzae hyphae, but do not affect resting conidia. The damage of the fungus is mediated, at least in part, by perforin. R. oryzae hyphae decrease the secretion of immunoregulatory molecules by NK cells, such as IFN-γ and RANTES, indicating an immunosuppressive effect of the fungus. Our data indicate that NK cells exhibit activity against Mucormycetes and future research should evaluate NK cells as a potential tool for adoptive immunotherapy in HSCT.

Aspergillus Fumigatus Activates Thrombocytes by Secretion of Soluble Compounds

The Journal of Infectious Diseases. Mar, 2013  |  Pubmed ID: 23225903

During invasive aspergillosis, platelets might be involved in immune defense, but they also might contribute to the pathology of the disease. We tested the hypothesis that Aspergillus secretes factors that influence the activity and functionality of thrombocytes. Platelets were incubated with medium wherein Aspergillus fumigatus was grown. This fungal culture supernatant potently stimulated thrombocytes in a time- and dose-dependent fashion, inducing release of alpha and dense granules, membrane alterations, aggregation, and formation of microparticles. Fungus-induced platelet activation could be confirmed in vivo: thrombocytes from mice infected with A. fumigatus showed a higher activation level than platelets from noninfected animals. Two stimulating components in the fungal culture supernatant were identified: a fungal serine protease and the mycotoxin gliotoxin. Activation of platelets by fungal factors stimulates antifungal functions: platelets gain the capacity to interact with foreign particles, and they become able to inhibit fungal growth, thus supporting the host immune network. However, some consequences of platelet activation might also be harmful, including excessive inflammation and induction of thrombosis. These findings imply that measuring platelet activation in patients might be an interesting diagnostic parameter.

Utility of PCR in Diagnosis of Invasive Fungal Infections: Real-life Data from a Multicenter Study

Journal of Clinical Microbiology. Mar, 2013  |  Pubmed ID: 23269732

Prospective studies addressing the clinical value of broad-range PCR using the internal transcribed spacer region (ITS) for diagnosis of microscopy-negative fungal infections in nonselected patient populations are lacking. We first assessed the diagnostic performance of ITS rRNA gene PCR compared with that of routine microscopic immunofluorescence examination. Second, we addressed prospectively the impact and clinical value of broad-range PCR for the diagnosis of infections using samples that tested negative by routine microscopy; the corresponding patients' data were evaluated by detailed medical record reviews. Results from 371 specimens showed a high concordance of >80% for broad-range PCR and routine conventional methods, indicating that the diagnostic performance of PCR for fungal infections is comparable to that of microscopy, which is currently considered part of the "gold standard." In this prospective study, 206 specimens with a negative result on routine microscopy were analyzed with PCR, and patients' clinical data were reviewed according to the criteria of 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. We found that broad-range PCR showed a sensitivity, specificity, positive predictive value, and negative predictive value of 57.1%, 97.0%, 80%, and 91.7%, respectively, for microscopy-negative fungal infections. This study defines a possible helpful role of broad-range PCR for diagnosis of microscopy-negative fungal infections in conjunction with other tests.

New Insights into Invasive Aspergillosis--from the Pathogen to the Disease

Current Pharmaceutical Design. 2013  |  Pubmed ID: 23278533

Disease manifestations with Aspergillus spp. are very diverse and dependent on interaction between the fungus and the host. Invasive aspergillosis (IA) is the most severe form of Aspergillus - associated disease found in immunocompromised hosts. Infections are mainly due to Aspergillus (A.) fumigatus, an air-borne opportunistic pathogen that causes 90% of IA. Mortality rate of this disease is still very high (50-95%), partly because of diagnostic difficulties, limited antifungal treatment options, weak conditions of patients at risk; but also in part because understanding of virulence factors involved in A. fumigatus pathogenicity and interactions of the pathogen with the host immune system is still poor. This review focuses on properties of A. fumigatus in terms of putative virulence factors and interactions of the pathogen with a main focus on the innate immune system.

Up-date on Diagnostic Strategies of Invasive Aspergillosis

Current Pharmaceutical Design. 2013  |  Pubmed ID: 23278540

Invasive fungal infections are a major cause of morbidity and mortality in immunosuppressed patients with fatality rates ranging from 30 % to 80 % in neutropenic patients. This results at least partly in difficulties obtaining a reliable and early diagnosis, followed by the fact that clinical symptoms are unspecific and of very limited use. Conventional methods may be difficult and are often delayed. Serological tests like the galactomannan enzyme immunoassay are presently most promising for diagnosing invasive aspergillosis, however, sensitivity and specificity vary within the studies. This review reflects the current situation of diagnosing invasive fungal infections with a special focus on how to best diagnose Aspergillus-related infections (e.g., culture, microscopy, imaging techniques, CT-guided biopsies). Promising molecular techniques under development will be discussed and their potential for routine diagnostic applications (e.g. lateral flow device, polymerase chain reaction [PCR]-based assays, rolling circle amplification [RCA], loop-mediated amplification [LAMP], nucleic acid sequence-based amplification [NASBA]). A summary is given on commercial assays (e.g., Platelia Aspergillus®, Fungitell®, in situ hybridization, immunohistochemisty) and their purposes as screening tools and/or diagnostic tools. This review gives a future outlook on how to best diagnose Aspergillus infections.

New Insight into Amphotericin B Resistance in Aspergillus Terreus

Antimicrobial Agents and Chemotherapy. Apr, 2013  |  Pubmed ID: 23318794

Amphotericin B (AMB) is the predominant antifungal drug, but the mechanism of resistance is not well understood. We compared the in vivo virulence of an AMB-resistant Aspergillus terreus (ATR) isolate with that of an AMB-susceptible A. terreus isolate (ATS) using a murine model for disseminated aspergillosis. Furthermore, we analyzed the molecular basis of intrinsic AMB resistance in vitro by comparing the ergosterol content, cell-associated AMB levels, AMB-induced intracellular efflux, and prooxidant effects between ATR and ATS. Infection of immunosuppressed mice with ATS or ATR showed that the ATS strain was more lethal than the ATR strain. However, AMB treatment improved the outcome in ATS-infected mice while having no positive effect on the animals infected with ATR. The in vitro data demonstrated that ergosterol content is not the molecular basis for AMB resistance. ATR absorbed less AMB, discharged more intracellular compounds, and had better protection against oxidative damage than the susceptible strain. Our experiments showed that ergosterol content plays a minor role in intrinsic AMB resistance and is not directly associated with intracellular cell-associated AMB content. AMB might exert its antifungal activity by oxidative injury rather than by an increase in membrane permeation.

Minireview: Host Defence in Invasive Aspergillosis

Mycoses. Jul, 2013  |  Pubmed ID: 23406508

Aspergillus is a saprophytic fungus, which mainly becomes pathogenic in immunosuppressed hosts. A failure of host defences results in a diverse set of illnesses, ranging from chronic colonisation, aspergilloma, invasive disease and hypersensitivity. A key concept in immune responses to Aspergillus species is that host susceptibility determines the morphological form, antigenic structure and physical location of the fungus. Traditionally, innate immunity has been considered as a first line of defence and activates adaptive immune mechanisms by the provision of specific signals; innate and adaptive immune responses are intimately linked. The T-helper cell (TH 1) response is associated with increased production of inflammatory cytokines IFN-γ, IL-2 and IL-12 and stimulation of antifungal effector cells. Alternatively, TH 2-type responses are associated with suppression of antifungal effector cell activity, decreased production of IFN-γ and increased concentrations of IL-4 and IL-10, which promote humoral responses to Aspergillus. The host's defensive capacity is defined by the sum of resistance and tolerance. Resistance displays the ability to limit fungal burden and elimination of the pathogen, and tolerance means the ability to limit host damage caused by immune response.

Invasive Pulmonary Mycosis Due to Penicillium Chrysogenum: a New Invasive Pathogen

Transplantation. Feb, 2013  |  Pubmed ID: 23423272

The Mediterranean Red Alga Asparagopsis Taxiformis Has Antifungal Activity Against Aspergillus Species

Mycoses. Sep, 2013  |  Pubmed ID: 23437896

The red algae Asparagopsis taxiformis collected from the Straits of Messina (Italy) were screened for antifungal activity against Aspergillus species. EUCAST methodology was applied and extracts showed antifungal activity against A. fumigatus, A. terreus and A. flavus. The lowest minimum inhibitory concentrations observed were <0.15 mg ml(-1) and the highest were >5 mg ml(-1) for Aspergillus spp. tested. Agar diffusion assays confirmed antifungal activity of A. taxiformis extracts in Aspergillus species.

Risk Factors and Outcomes in Lung Transplant Recipients with Nodular Invasive Pulmonary Aspergillosis

The Journal of Infection. Jul, 2013  |  Pubmed ID: 23567625

Whether nodular lesions have specific risk-factors or influence outcomes in lung transplant recipients with invasive aspergillosis, is not fully known.

Clinical-scale Generation of Multi-specific Anti-fungal T Cells Targeting Candida, Aspergillus and Mucormycetes

Cytotherapy. Mar, 2013  |  Pubmed ID: 23579059

Invasive fungal infections, in particular, infections caused by Candida, Aspergillus and mucormycetes, are a major cause of morbidity and mortality in patients undergoing allogeneic hematopoietic stem cell transplantation. Adoptive transfer of donor-derived anti-fungal T cells shows promise to restore immunity and to offer a cure. Because T cells recognize only specific epitopes, the low rate of patients in which the causal fungal pathogen can be identified and the considerable number of patients with co-infection with several genera or species of fungi significantly limit the application of adoptive immunotherapy.

Combined Antifungal Approach for the Treatment of Invasive Mucormycosis in Patients with Hematologic Diseases: a Report from the SEIFEM and FUNGISCOPE Registries

Haematologica. Oct, 2013  |  Pubmed ID: 23716556

Virulence and Thrombocyte Affectation of Two Aspergillus Terreus Isolates Differing in Amphotericin B Susceptibility

Medical Microbiology and Immunology. Oct, 2013  |  Pubmed ID: 23722593

Aspergillus terreus-induced invasive infections exhibit high lethality, partly due to the intrinsic resistance for amphotericin B (AmB). We compared the virulence and pathogenesis of an AmB-resistant isolate of A. terreus (ATR) with that of a rare variant showing enhanced sensitivity for AMB (ATS). The modifications that result in enhanced AmB sensitivity of isolates are not associated with reduced virulence in vivo; instead, the ATS-infected mice died even faster than the ATR-infected animals. Since A. terreus enters the blood stream in most patients and frequently induces thrombosis, we studied a putative correlation between virulence of the two A. terreus isolates and their effect on thrombocytes. Those mice infected with the more virulent ATS isolate had lower thrombocyte numbers and more phosphatidylserine exposure on platelets than ATR-infected mice. In vitro experiments confirmed that ATS and ATR differ in their effect on thrombocytes. Conidia, aleurioconidia and hyphae of ATS were more potent than ATR to trigger thrombocyte stimulation, and thrombocytes adhered better to ATS than to ATR fungal structures. Furthermore, ATS secreted more soluble factors that triggered platelet stimulation than ATR. Thus, it might be suggested that the capacity of a fungal isolate to modulate thrombocyte parameters contributes to its virulence in vivo.

Rep-PCR and RAPD-PCR Fingerprinting of Aspergillus Terreus

Medical Mycology. Nov, 2013  |  Pubmed ID: 23758105

We compared two PCR methods for molecular typing the medically important filamentous fungus Aspergillus terreus. In a set of 46 strains investigated we found 19 and 12 different fingerprinting types obtained by random amplified polymorphic DNA PCR (RAPD) and semi-automated repetitive element PCR (rep-PCR), respectively.

Determination of Isavuconazole Susceptibility of Aspergillus and Candida Species by the EUCAST Method

Antimicrobial Agents and Chemotherapy. Nov, 2013  |  Pubmed ID: 23959309

Isavuconazole is a novel expanded-spectrum triazole, which has recently been approved by the FDA as an orphan drug to treat invasive aspergillosis and is currently being studied in phase III clinical trials for invasive candidiasis. The susceptibility of relatively few clinical isolates has been reported. In this study, the isavuconazole susceptibilities of 1,237 Aspergillus and 2,010 Candida geographically diverse clinical isolates were determined by EUCAST methodology at four European mycology laboratories, producing the largest multicenter data set thus far for this compound. In addition, a blinded collection of 30 cyp51A mutant Aspergillus fumigatus clinical isolates and 10 wild-type isolates was tested. From these two data sets, the following preliminary epidemiological cutoff (ECOFF) values were suggested: 2 mg/liter for Aspergillus fumigatus, Aspergillus terreus, and Aspergillus flavus; 4 mg/liter for Aspergillus niger; 0.25 mg/liter for Aspergillus nidulans; and 0.03 mg/liter for Candida albicans, Candida parapsilosis, and Candida tropicalis. Unfortunately, ECOFFs could not be determined for Candida glabrata or Candida krusei due to an unexplained interlaboratory MIC variation. For the blinded collection of A. fumigatus isolates, all MICs were ≤2 mg/liter for wild-type isolates. Differential isavuconazole MICs were observed for triazole-resistant A. fumigatus isolates with different cyp51A alterations: TR34/L98H mutants had elevated isavuconazole MICs, whereas isolates with G54 and M220 alterations had MICs in the wild-type range, suggesting that the efficacy of isavuconazole may not be affected by these alterations. This study will be an aid in interpreting isavuconazole MICs for clinical care and an important step in the future process of setting official clinical breakpoints.

Immune Reconstitution Syndrome-like Entity in Lung Transplant Recipients with Invasive Aspergillosis

Transplant Immunology. Dec, 2013  |  Pubmed ID: 24076039

Incidence, characteristics, and risk-factors for invasive aspergillosis (IA)-associated immune reconstitution syndrome (IRS) in lung transplant recipients are not known.

Hypoxia Promotes Danger-mediated Inflammation Via Receptor for Advanced Glycation End Products in Cystic Fibrosis

American Journal of Respiratory and Critical Care Medicine. Dec, 2013  |  Pubmed ID: 24127697

Hypoxia regulates the inflammatory-antiinflammatory balance by the receptor for advanced glycation end products (RAGE), a versatile sensor of damage-associated molecular patterns. The multiligand nature of RAGE places this receptor in the midst of chronic inflammatory diseases.

Platelets As Immune Cells in Infectious Diseases

Future Microbiology. Nov, 2013  |  Pubmed ID: 24199802

Platelets have been shown to cover a broad range of functions. Besides their role in hemostasis, they have immunological functions and thus participate in the interaction between pathogens and host defense. Platelets have a broad repertoire of receptor molecules that enable them to sense invading pathogens and infection-induced inflammation. Consequently, platelets exert antimicrobial effector mechanisms, but also initiate an intense crosstalk with other arms of the innate and adaptive immunity, including neutrophils, monocytes/macrophages, dendritic cells, B cells and T cells. There is a fragile balance between beneficial antimicrobial effects and detrimental reactions that contribute to the pathogenesis, and many pathogens have developed mechanisms to influence these two outcomes. This review aims to highlight aspects of the interaction strategies between platelets and pathogenic bacteria, viruses, fungi and parasites, in addition to the subsequent networking between platelets and other immune cells, and the relevance of these processes for the pathogenesis of infections.

Why is Biopsy of Suspected Fungal Lung Lesions Necessary?

Medical Mycology Case Reports. Aug, 2013  |  Pubmed ID: 24432240

The recognition of antifungal resistance is necessary for the choice of the appropriate treatment in patients with invasive fungal disease. In this case report, the need for a computed tomography-guided percutaneous lung biopsy of a suspected fungal lesion in a patient treated for acute leukemia is demonstrated. Detection of Amphothericin-B resistant Aspergillus flavus infection has prompted the switch in antifungal therapy, followed by full resolution of symptoms, completion of chemotherapy and remission since then.

Breakpoints for Antifungal Agents: an Update from EUCAST Focussing on Echinocandins Against Candida Spp. and Triazoles Against Aspergillus Spp

Drug Resistance Updates : Reviews and Commentaries in Antimicrobial and Anticancer Chemotherapy. Dec, 2013  |  Pubmed ID: 24618110

Candida and Aspergillus infections have emerged as significant pathogens in recent decades. During this same time, broad spectrum triazole and echinocandin antifungal agents have been developed and increasingly used. One consequence of widespread use is leading to the emergence of mutants with acquired resistance mutations. Therefore, accurate susceptibility testing and appropriate clinical breakpoints for the interpretation of susceptibility results have become increasingly important. Here we review the underlying methodology by which breakpoints have been selected by EUCAST (European Committee on Antimicrobial Susceptibility Testing). Five parameters are evaluated: dosing regimens used; EUCAST MIC distributions from multiple laboratories, species and compound specific epidemiological cut off values (upper MIC limits of wild type isolates or ECOFFs), pharmacokinetic/pharmacodynamic relationships and targets associated with outcome and finally clinical data by species and MIC when available. The general principles are reviewed followed by a detailed review of the individual aspects for Candida species and the three echinocandins and for Aspergillus and the three mould-active azoles. This review provides an update of the subcommittee on antifungal susceptibility testing (AFST) of the EUCAST methodology and summarises the current EUCAST breakpoints for Candida and Aspergillus. Recommendations about applicability of antifungal susceptibility testing in the routine setting are also included.

EUCAST Technical Note on Candida and Micafungin, Anidulafungin and Fluconazole

Mycoses. Jun, 2014  |  Pubmed ID: 24417759

The European Committee on Antimicrobial Susceptibility Testing Subcommittee on Antifungal Susceptibility Testing has determined breakpoints for micafungin and revised breakpoints for anidulafungin and fluconazole for Candida spp. This Technical Note is based on the corresponding rationale documents (http://www.eucast.org). The micafungin breakpoints are based on PK data, animal PK/PD data, microbiological data and clinical experience. The anidulafungin breakpoints for C. parapsilosis and fluconazole breakpoints for C. glabrata have been modified to species-specific values that categorise the wild-type as intermediate to accommodate use of these compounds in some clinical situations.

Risk Assessment of Transfusion-associated Babesiosis in Tyrol: Appraisal by Seroepidemiology and Polymerase Chain Reaction

Transfusion. Jul, 2014  |  Pubmed ID: 24673158

After malaria, babesiosis is the second most common transfusion-transmitted parasitic disease in the United States. In Europe, one reported transfusion case, concerning Babesia microti, occurred in Germany.

In Vitro Antifungal Susceptibility of Candida Glabrata to Caspofungin and the Presence of FKS Mutations Correlate with Treatment Response in an Immunocompromised Murine Model of Invasive Infection

Antimicrobial Agents and Chemotherapy. Jul, 2014  |  Pubmed ID: 24733474

It has been argued that the in vitro activity of caspofungin (CSP) is not a good predictor of the outcome of echinocandin treatment in vivo. We evaluated the in vitro activity of CSP and the presence of FKS mutations in the hot spot 1 (HS1) region of the FKS1 and FKS2 genes in 17 Candida glabrata strains with a wide range of MICs. The efficacy of CSP against systemic infections from each of the 17 strains was evaluated in a murine model. No HS1 mutations were found in the eight strains showing MICs for CSP of ≤ 0.5 μg/ml, but they were present in eight of the nine strains with MICs of ≥ 1 μg/ml, i.e., three in the FKS1 gene and five in the FKS2 gene. CSP was effective for treating mice infected with strains with MICs of ≤ 0.5 μg/ml, showed variable efficacy in animals challenged with strains with MICs of 1 μg/ml, and did not work in those with strains with MICs of >1 μg/ml. In addition, mutations, including one reported for the first time, were found outside the HS1 region in the FKS2 gene of six strains with different MICs, but their presence did not influence drug efficacy. The in vitro activity of CSP was compared with that of another echinocandin, anidulafungin, suggesting that the MICs of both drugs, as well as mutations in the HS1 regions of the FKS1 and FKS2 genes, are predictive of outcome.

Computed Tomography Guided Percutaneous Lung Biopsies and Suspected Fungal Infections in Pediatric Cancer Patients

Pediatric Blood & Cancer. Sep, 2014  |  Pubmed ID: 24803392

The spectrum of potential fungal pathogens known to cause invasive pulmonary infections has grown as a result of intensified immunosuppressive therapy and the emergence of antifungal resistance.

Bronchoalveolar Lavage Lateral-flow Device Test for Invasive Pulmonary Aspergillosis in Solid Organ Transplant Patients: a Semiprospective Multicenter Study

Transplantation. Oct, 2014  |  Pubmed ID: 24879383

Invasive pulmonary aspergillosis (IPA) remains an important cause of morbidity and mortality among patients undergoing solid organ transplantation (SOT). Because of the crude mortality of 80% to 90% in the absence of adequate treatment, timely diagnosis and early intervention with antifungal drugs are key factors in the successful treatment of IPA. Diagnosis, however, remains difficult. Therefore, new diagnostic tests are urgently needed. The Lateral-Flow Device (LFD) test is a rapid (15 min) single-sample point-of-care test that is based on the detection of an Aspergillus extracellular glycoprotein antigen by monoclonal antibody JF5.

Laboratory Diagnosis of Mucormycosis: Current Status and Future Perspectives

Future Microbiology. 2014  |  Pubmed ID: 24957094

Fungal infections caused by members of the Mucorales order are rapidly progressing and fatal. The importance of mucormycosis has grown in recent years as the number of patients with predisposing factors has increased dramatically. Clinical symptoms are elusive and conventional techniques are often insensitive and unspecific; in particular, cultures are often negative even though direct microscopy is positive. For early diagnosis of the causative agent of disease and subsequently guiding therapy to improving patients' outcome, molecular assays are promising add-ons. This article provides an overview on current laboratory methods for diagnosing mucormycosis with a special focus on new molecular-based tools. We aim to highlight the pros and cons of various techniques at hand. Given the increase in number and the severity of these infections, molecular approaches for improved diagnosis are highly warranted.

The Viral Make-up Makes a World of Difference

AIDS Research and Human Retroviruses. Jul, 2014  |  Pubmed ID: 24984236

Assessing Micafungin/triazole Combinations for the Treatment of Invasive Scedosporiosis Due to Scedosporium Apiospermum and Scedosporium Boydii

The Journal of Antimicrobial Chemotherapy. Nov, 2014  |  Pubmed ID: 24986494

Scedosporium infections are associated with high therapeutic failure rates. Combination therapy may be an alternative approach to improve outcome. The in vitro and in vivo efficacy of micafungin plus posaconazole or plus voriconazole was investigated herein.

Platelet Immunology in Fungal Infections

Thrombosis and Haemostasis. Oct, 2014  |  Pubmed ID: 24990293

Up to date, perception of platelets has changed from key players in coagulation to multitaskers within the immune network, connecting its most diverse elements and crucially shaping their interplay with invading pathogens such as fungi. In addition, antimicrobial effector molecules and mechanisms in platelets enable a direct inhibitory effect on fungi, thus completing their immune capacity. To precisely assess the impact of platelets on the course of invasive fungal infections is complicated by some critical parameters. First, there is a fragile balance between protective antimicrobial effects and detrimental reactions that aggravate the fungal pathogenesis. Second, some platelet effects are exerted indirectly by other immune mediators and are thus difficult to quantify. Third, drugs such as antimycotics, antibiotics, or cytostatics, are commonly administered to the patients and might modulate the interplay between platelets and fungi. Our article highlights selected aspects of the complex interactions between platelets and fungi and the relevance of these processes for the pathogenesis of fungal infections.

Incidence of Cyp51 A Key Mutations in Aspergillus Fumigatus-a Study on Primary Clinical Samples of Immunocompromised Patients in the Period of 1995-2013

PloS One. 2014  |  Pubmed ID: 25072733

As the incidence of azole resistance in Aspergillus fumigatus is rising and the diagnosis of invasive aspergillosis (IA) in immunocompromised patients is rarely based on positive culture yield, we screened our Aspergillus DNA sample collection for the occurrence of azole resistance mediating cyp51 A key mutations. Using two established, a modified and a novel polymerase chain reaction (PCR) assays followed by DNA sequence analysis to detect the most frequent mutations in the A. fumigatus cyp51 A gene conferring azole resistance (TR34 (tandem repeat), L98H and M220 alterations). We analyzed two itraconazole and voriconazole and two multi-azole resistant clinical isolates and screened 181 DNA aliquots derived from clinical samples (blood, bronchoalveolar lavage (BAL), biopsies, cerebrospinal fluid (CSF)) of 155 immunocompromised patients of our Aspergillus DNA sample collection, previously tested positive for Aspergillus DNA and collected between 1995 and 2013. Using a novel PCR assay for the detection of the cyp51 A 46 bp tandem repeat (TR46) directly from clinical samples, we found the alteration in a TR46/Y121F/T289A positive clinical isolate. Fifty stored DNA aliquots from clinical samples were TR46 negative. DNA sequence analysis revealed a single L98H mutation in 2010, two times the L98H alteration combined with TR34 in 2011 and 2012 and a so far unknown N90K mutation in 1998. In addition, four clinical isolates were tested positive for the TR34/L98H combination in the year 2012. We consider our assay of epidemiological relevance to detect A. fumigatus azole resistance in culture-negative clinical samples of immunocompromised patients; a prospective study is ongoing.

EUCAST Testing of Isavuconazole Susceptibility in Aspergillus: Comparison of Results for Inoculum Standardization Using Conidium Counting Versus Optical Density

Antimicrobial Agents and Chemotherapy. Nov, 2014  |  Pubmed ID: 25136005

The EUCAST E.DEF9.1 standard recommends standardization of the inoculum concentration by conidium counting using a hemocytometer rather than a spectrophotometer. In this study, we investigated whether the choice of these methods influenced isavuconazole MICs. A blinded collection of 30 molecularly characterized azole-resistant isolates and 10 wild-type Aspergillus fumigatus isolates was shared with four different laboratories. Additionally, each laboratory selected approximately 100 A. fumigatus isolates and 50 isolates each of A. flavus, A. nidulans, A. niger, and A. terreus (1,237 isolates in total). Three laboratories (laboratories 1 to 3) used conidium counting. One laboratory standardized the inoculum using a spectrophotometer (that is, by use of the optical density [OD]) and is referred to as the OD laboratory. Correlation coefficients, intraclass correlation coefficients, and essential agreement were calculated, and 2-log-unit differences were assessed (paired t test). The MIC range for the blinded collection was 0.25 to 16 mg/liter, and a 1-dilution-step difference between the MIC50 and MIC90 across the four laboratories was detected and a 2-dilution-step difference between the modal MICs was detected. Compared to the results for laboratories 1 and 2, a significant correlation was found for the OD laboratory MIC data (correlation coefficients, 0.85 and 0.93, respectively; intraclass correlation coefficients, 0.88 and 0.96, respectively). The number of mutant isolates whose MICs overlapped those of the wild-type isolates was the lowest for the OD laboratory (14/30 [46.7%] mutant isolates), whereas the numbers were 18/30 (60%) isolates for laboratory 1, 17/30 (56.7%) isolates for laboratory 2, and 21/30 (70%) isolates for laboratory 3. For the A. flavus, A. fumigatus, A. nidulans, A. niger, and A. terreus isolates, comparative analysis again defined the MIC distributions from the OD laboratory to be in excellent agreement with those from laboratories 1 and 2 across all five Aspergillus spp. The findings suggest that EUCAST testing using OD determination is an appropriate alternative for standardization of Aspergillus inoculum concentrations.

De Novo Whole-Genome Sequence and Genome Annotation of Lichtheimia Ramosa

Genome Announcements. Sep, 2014  |  Pubmed ID: 25212617

We report the annotated draft genome sequence of Lichtheimia ramosa (JMRC FSU:6197). It has been reported to be a causative organism of mucormycosis, a rare but rapidly progressive infection in immunocompromised humans. The functionally annotated genomic sequence consists of 74 scaffolds with a total number of 11,510 genes.

Primary Antifungal Prophylaxis with Micafungin in Patients with Haematological Malignancies: Real-life Data from a Retrospective Single-centre Observational Study

European Journal of Haematology. Mar, 2015  |  Pubmed ID: 25082655

Mould-active antifungal prophylaxis is increasingly used in patients at risk for invasive fungal disease. Between June 2011 and June 2012, one hundred patients with various haematological malignancies at risk for invasive fungal disease received primary antifungal prophylaxis with intravenous micafungin at a daily dosage of 50 mg during neutropenia. The median number of days on micafungin prophylaxis was 14 (range, 6-48 d). The incidence of proven and probable breakthrough invasive fungal diseases (bIFDs) was 6% and 3%, respectively. There were two bloodstream infections caused by yeasts or yeast-like fungi (Candida krusei, Trichosporon asahii) in two patients during the neutropenic phase after allogeneic haematopoietic stem cell transplantation. Four proven bIFDs caused by non-Aspergillus moulds and three cases of probable pulmonary bIFDs were documented during the neutropenic phase after induction/consolidation chemotherapy for acute leukaemia. Colonisation with Candida spp. was documented in 51% of the patients with none of the isolates being in vitro micafungin resistant. Compared to a historical control, receiving primary prophylaxis with posaconazole micafungin is at least as effective in preventing IFD. In both cohorts, bIFDs were exclusively caused by emerging pathogens with a highly preserved in vitro sensitivity to amphotericin B.

Susceptibility Profiles of Amphotericin B and Posaconazole Against Clinically Relevant Mucorales Species Under Hypoxic Conditions

Antimicrobial Agents and Chemotherapy. Feb, 2015  |  Pubmed ID: 25451049

The effect of hypoxic conditions on the in vitro efficacy of amphotericin B and posaconazole against Mucorales was evaluated by defining MICs with Etest and broth microdilution and identifying minimal fungicidal concentrations (MFCs). With Etest, oxygen-dependent changes were detected, while the MIC and the MFC determined with broth microdilution remained unaltered with reduced oxygen levels. The observed differences depended on the method used.

Effect of Reduced Oxygen on the Antifungal Susceptibility of Clinically Relevant Aspergilli

Antimicrobial Agents and Chemotherapy. Mar, 2015  |  Pubmed ID: 25547350

The influence of hypoxia on the in vitro activities of amphotericin B, azoles, and echinocandins against Aspergillus spp. was evaluated by comparing MICs, minimal fungicidal concentrations (MFCs), and epidemiological cutoffs (ECOFFs). Changes of MIC distributions due to hypoxia largely depend on the method, the species, and the growth ability under hypoxia. The activities of antifungals were not significantly altered under hypoxia, except for Aspergillus terreus, for which the activity changed from fungicidal to fungistatic.

Immediate T-Helper 17 Polarization Upon Triggering CD11b/c on HIV-Exposed Dendritic Cells

The Journal of Infectious Diseases. Jul, 2015  |  Pubmed ID: 25583169

Early on in human immunodeficiency virus (HIV) type 1 infection, gut T-helper (Th) 17 cells are massively depleted leading eventually to compromised intestinal barrier function and excessive immune activation. In contrast, the functional Th17 cell compartment of the gut is well-maintained in nonpathogenic simian immunodeficiency virus infection as well as HIV-1 long-term nonprogressors. Here, we show that dendritic cells (DCs) loaded with HIV-1 bearing high surface complement levels after incubation in plasma from HIV-infected individuals secreted significantly higher concentrations of Th17-polarizing cytokines than DCs exposed to nonopsonized HIV-1. The enhanced Th17-polarizing capacity of in vitro-generated and BDCA-1(+) DCs directly isolated from blood was linked to activation of ERK. In addition, C3a produced from DCs exposed to complement-opsonized HIV was associated with the higher Th17 polarization. Our in vitro and ex vivo data, therefore, indicate that complement opsonization of HIV-1 strengthens DC-mediated antiviral immune functions by simultaneously triggering Th17 expansion and intrinsic C3 formation via DC activation.

Differential Gene Expression in Aspergillus Fumigatus Induced by Human Platelets in Vitro

International Journal of Medical Microbiology : IJMM. May, 2015  |  Pubmed ID: 25661519

Invasive aspergillosis is characterized by vascular invasion and thrombosis. In order to determine the antifungal activity of human platelets, hyphal elongation and metabolic activity of a clinical A. fumigatus isolate were measured. Genome-wide identification of differentially expressed genes in A. fumigatus was performed after exposure to platelets for 15, 30, 60 and 180 min. Data were analyzed by gene ontology annotation as well as functional categories (FunCat) and KEGG enrichment analyses. Platelets attenuated hyphal elongation and viability of A. fumigatus and in total 584 differentially expressed genes were identified, many of which were associated with regulation of biological processes, stress response, transport and metabolism. FunCat and KEGG enrichment analyses showed stress response and metabolic adaptation to be increased in response to platelets. Our findings demonstrate that A. fumigatus displayed a specific transcriptional response when exposed to platelets, thus reflecting their antifungal activities.

Bronchoalveolar Lavage Fluid (1,3)β-D-Glucan for the Diagnosis of Invasive Fungal Infections in Solid Organ Transplantation: A Prospective Multicenter Study

Transplantation. Sep, 2015  |  Pubmed ID: 25710608

Prompt diagnosis of invasive fungal infections (IFI) remains a challenge. (1,3)β-D-glucan detection in bronchoalveolar lavage (BAL) fluid by Fungitell assay aims to further improve upon the test's utility by directly applying it to specimens from the target organ.

Necrotizing Mycosis Due to Verruconis Gallopava in an Immunocompetent Patient

Infection. Dec, 2015  |  Pubmed ID: 25744338

Verruconis gallopava is a dematiaceous mould usually causing saprophytic infection in immunosuppressed host. Only a few cases have been published even in immunocompromised states. We present a rare case of pulmonary involvement in an immunocompetent patient with recurrent disease. The mid-aged woman had no evidence of any disease causing impaired immune response. Recurrent disease shows pulmonary infiltrates and symptoms of allergic bronchopulmonary mycosis. We describe an emerging pathogen that has been found in an immunocompetent host. Eradication was not possible despite the use of several different antifungal drugs. Further recurrence of infection in the described patient is probable.

Identification of Aspergillus Fumigatus Surface Components That Mediate Interaction of Conidia and Hyphae With Human Platelets

The Journal of Infectious Diseases. Oct, 2015  |  Pubmed ID: 25810442

Platelets were recently identified as a part of innate immunity. They are activated by contact with Aspergillus fumigatus; putative consequences include antifungal defense but also thrombosis, excessive inflammation, and thrombocytopenia. We aimed to identify those fungal surface structures that mediate interaction with platelets.

In Vitro Activities of Amphotericin B, Terbinafine, and Azole Drugs Against Clinical and Environmental Isolates of Aspergillus Terreus Sensu Stricto

Antimicrobial Agents and Chemotherapy. 2015  |  Pubmed ID: 25824228

The antifungal susceptibilities of 40 clinical and environmental isolates of A. terreus sensu stricto to amphotericin B, terbinafine, itraconazole, and voriconazole were determined in accordance with CLSI document M38-A2. All isolates had itraconazole and voriconazole MICs lower than epidemiologic cutoff values, and 5% of the isolates had amphotericin B MICs higher than epidemiologic cutoff values. Terbinafine showed the lowest MICs. No significant differences were found when MICs of clinical and environmental isolates were compared.

Etest Cannot Be Recommended for in Vitro Susceptibility Testing of Mucorales

Antimicrobial Agents and Chemotherapy. 2015  |  Pubmed ID: 25845881

Amphotericin B and posaconazole susceptibility patterns were determined for the most prevalent Mucorales, following EUCAST (European Committee on Antimicrobial Susceptibility Testing) broth microdilution guidelines. In parallel, Etest was performed and evaluated against EUCAST. The overall agreement of MICs gained with Etest and EUCAST was 75.1%; therefore, Etest cannot be recommended for antifungal susceptibility testing of Mucorales. Amphotericin B was the most active drug against Mucorales species in vitro, while the activities of posaconazole were more restricted.

Blocking Hsp70 Enhances the Efficiency of Amphotericin B Treatment Against Resistant Aspergillus Terreus Strains

Antimicrobial Agents and Chemotherapy. Jul, 2015  |  Pubmed ID: 25870060

The polyene antifungal amphotericin B (AmB) is widely used to treat life-threatening fungal infections. Even though AmB resistance is exceptionally rare in fungi, most Aspergillus terreus isolates exhibit an intrinsic resistance against the drug in vivo and in vitro. Heat shock proteins perform a fundamental protective role against a multitude of stress responses, thereby maintaining protein homeostasis in the organism. In this study, we elucidated the role of heat shock protein 70 (Hsp70) family members and compared resistant and susceptible A. terreus clinical isolates. The upregulation of cytoplasmic Hsp70 members at the transcriptional as well as translational levels was significantly higher with AmB treatment than without AmB treatment, particularly in resistant A. terreus isolates, thereby indicating a role of Hsp70 proteins in the AmB response. We found that Hsp70 inhibitors considerably increased the susceptibility of resistant A. terreus isolates to AmB but exerted little impact on susceptible isolates. Also, in in vivo experiments, using the Galleria mellonella infection model, cotreatment of resistant A. terreus strains with AmB and the Hsp70 inhibitor pifithrin-μ resulted in significantly improved survival compared with that achieved with AmB alone. Our results point to an important mechanism of regulation of AmB resistance by Hsp70 family members in A. terreus and suggest novel drug targets for the treatment of infections caused by resistant fungal isolates.

Complement and Platelets: Mutual Interference in the Immune Network

Molecular Immunology. Sep, 2015  |  Pubmed ID: 25886718

In recent years, the view of platelets has changed from mere elements of hemostasis to immunological multitaskers. They are connected in manifold ways to other cellular and humoral components of the immune network, one of which is the complement system, a potent player in soluble innate immunity. Our article reviews the crucial and complex interplay between platelets and complement, focusing on mutual regulation of these two interaction partners by their respective molecular mechanisms. Furthermore, the putative relevance of these processes to infectious diseases, inflammatory conditions, and autoimmune disorders, as well as the treatment of patients with biomaterials is highlighted.

Aspergillus-Specific Lateral-Flow Device and Real-Time PCR Testing of Bronchoalveolar Lavage Fluid: a Combination Biomarker Approach for Clinical Diagnosis of Invasive Pulmonary Aspergillosis

Journal of Clinical Microbiology. Jul, 2015  |  Pubmed ID: 25903568

Clinical experience with the impact of serum biomarkers for invasive fungal disease (IFD) varies markedly in hemato-oncology. Invasive pulmonary aspergillosis (IPA) is the most common manifestation, so we evaluated biomarkers in bronchoalveolar lavage (BAL) fluid. An Aspergillus-specific lateral-flow device (LFD), quantitative real-time PCR (qPCR), and the galactomannan (GM) test were used with 32 BAL fluid samples from 32 patients at risk of IPA. Eight patients had proven IPA, 3 had probable IPA, 6 had possible IPA, and 15 patients had no IPA by European Organization for Research and Treatment of Cancer Invasive Fungal Infections Cooperative Group/Mycoses Study Group of the National Institute of Allergy and Infectious Diseases (EORTC/MSG) criteria. The diagnostic accuracies of the tests were evaluated, and pairwise agreement between biomarkers was calculated. The diagnostic performance of the EORTC/MSG criteria was evaluated against the test(s) identified to be the most useful for IPA diagnosis. Using the EORTC/MSG criteria, the sensitivities of qPCR and LFD were 100% and the sensitivity of the GM test was 87.5% (GM test index cutoff, >0.8), with the tests having specificities of between 66.7 and 86.7%. The agreement between the results of qPCR and LFD was almost perfect (Cohen's kappa coefficient = 0.93, 95% confidence interval, 0.81 to 1.00). LFD and qPCR combined had a sensitivity of 100% and a specificity of 85.7%. Calcofluor staining and culture of all BAL fluid samples were negative for fungal infection. The median time from the start of mold-active antifungal therapy to the time of collection of BAL fluid was 6 days. Reversing roles and using dual testing by LFD and qPCR to classify cases, the EORTC/MSG criteria had a sensitivity of 83.3%. All three tests are useful for the diagnosis of IPA in BAL fluid samples. Despite the significant delays between the start of antifungal therapy and bronchoscopy, unlike microscopy and culture, the biomarkers remained informative. In particular, the combination of LFD and qPCR allows the sensitive and specific detection of IPA.

Multicenter Evaluation of a Lateral-flow Device Test for Diagnosing Invasive Pulmonary Aspergillosis in ICU Patients

Critical Care (London, England). Apr, 2015  |  Pubmed ID: 25927915

The incidence of invasive pulmonary aspergillosis (IPA) in intensive care unit (ICU) patients is increasing, and early diagnosis of the disease and treatment with antifungal drugs is critical for patient survival. Serum biomarker tests for IPA typically give false-negative results in non-neutropenic patients, and galactomannan (GM) detection, the preferred diagnostic test for IPA using bronchoalveolar lavage (BAL), is often not readily available. Novel approaches to IPA detection in ICU patients are needed. In this multicenter study, we evaluated the performance of an Aspergillus lateral-flow device (LFD) test for BAL IPA detection in critically ill patients.

Advances in the Management of Fungal Infections

Mycoses. Jun, 2015  |  Pubmed ID: 26033250

Antifungal Drug Resistance Among Candida Species: Mechanisms and Clinical Impact

Mycoses. Jun, 2015  |  Pubmed ID: 26033251

The epidemiology of Candida infections has changed in recent years. Although Candida albicans is still the main cause of invasive candidiasis in most clinical settings, a substantial proportion of patients is now infected with non-albicans Candida species. The various Candida species vary in their susceptibility to the most commonly used antifungal agents, and the intrinsic resistance to antifungal therapy seen in some species, along with the development of acquired resistance during treatment in others, is becoming a major problem in the management of Candida infection. A better understanding of the mechanisms and clinical impact of antifungal drug resistance is essential for the efficient treatment of patients with Candida infection and for improving treatment outcomes. Herein, we report resistance to the azoles and echinocandins among Candida species.

Amphotericin B Resistance in Aspergillus Terreus Is Overpowered by Coapplication of Pro-oxidants

Antioxidants & Redox Signaling. Dec, 2015  |  Pubmed ID: 26054424

Invasive fungal infections have significantly increased over the past decades in immunocompromised individuals and high-risk patients. Amphotericin B (AmB) exerts a powerful and broad activity against a vast array of fungi and has a remarkably low rate of microbial resistance. However, most isolates of Aspergillus terreus developed an intrinsic resistance against AmB, and during this study, we characterized the mode of action of this polyene antifungal drug in more detail in resistant (ATR) and rare susceptible (ATS) clinical isolates of A. terreus.

Diagnostic Accuracy of the Aspergillus-specific Bronchoalveolar Lavage Lateral-flow Assay in Haematological Malignancy Patients

Mycoses. Aug, 2015  |  Pubmed ID: 26103209

We evaluated the performance of the Aspergillus-specific lateral-flow device (LFD) test for diagnosing invasive pulmonary aspergillosis (IPA) in patients with underlying haematological malignancies. Participating centres were the two Austrian University Hospitals of Graz and Innsbruck. LFD performance was evaluated with 95 bronchoalveolar lavage fluid (BALF) samples from 72 patients collected prospectively in Graz, and with 24 BALF bio bank samples from 23 patients (21 samples with probable IPA) in Innsbruck. Invasive fungal infections were classified according to the revised European Organization of Research and Treatment of Cancer/Mycoses Study Group criteria. Overall, 27 patients (30 samples) had probable IPA, 32 (43 samples) possible and 36 (46 samples) did not fulfil IPA criteria. The vast majority of patients - in particular those with probable IPA - received mould-active treatment before bronchoscopy. Sensitivity, specificity, positive predictive value and negative-predictive-value for probable IPA diagnosis using the BALF-LFD test were 71%, 76%, 35% and 94% for the Graz cohort. Sensitivity of the BALF-LFD test for probable IPA was 57% in Innsbruck bio bank samples. Our results indicate that the BALF-LFD-test provides fast results with moderate sensitivities in patients with underlying haematological malignancies. Similar to other diagnostic tests and biomarkers sensitivity of the test may be influenced by ongoing systemic mould-active treatment.

Galleria Mellonella As a Host Model to Study Aspergillus Terreus Virulence and Amphotericin B Resistance

Virulence. 2015  |  Pubmed ID: 26107350

The aim of this study was to investigate if the alternative in vivo model Galleria mellonella can be used (i) to determine differences in pathogenicity of amphotericin B (AMB) resistant and susceptible A. terreus isolates, (ii) to evaluate AMB efficacy in vivo (iii) and to correlate outcome to in vitro susceptibility data. Larvae were infected with 2 A. terreus AMB resistant (ATR) and 3 AMB susceptible (ATS) isolates and survival rates were correlated to physiological attributes and killing ability of larval haemocytes. Additionally, infected larvae were treated with different concentrations of L-AMB. Haemocyte density were ascertained to evaluate the influence of L-AMB on the larval immune cells. Larvae were sensitive to A. terreus infection in an inoculum-size and temperature dependent manner. In vitro susceptibility to L-AMB correlated with in vivo outcome of antifungal treatment, defining an AMB susceptible strain cluster of A. terreus. Susceptibility to L-AMB increased virulence potential in the larval model, but this increase was also in accordance with faster growth and less damage caused by larval haemocytes. L-AMB treatment primed the larval immune response by increasing haemocyte density. G. mellonella provides a convenient model for the in vivo screening of A. terreus virulence and treatment options, contributing to the generation of a hypothesis that can be further tested in refined experiments in mammalian models.

Biliary Amphotericin B Pharmacokinetics and Pharmacodynamics in Critically Ill Liver Transplant Recipients Receiving Treatment with Amphotericin B Lipid Formulations

International Journal of Antimicrobial Agents. Sep, 2015  |  Pubmed ID: 26119497

Fungal cholangitis is a potentially life-threatening condition. As amphotericin B (AmB) has a broad antimycotic spectrum, in this study its biliary penetration and activity was determined in two patients treated with liposomal AmB (L-AmB) and in one patient receiving AmB colloidal dispersion (ABCD). Biliary and plasma AmB levels were quantified by high-performance liquid chromatography after purification by solid-phase extraction. For assessment of biliary AmB activity, isolates of Candida albicans, Candida tropicalis, Candida glabrata and Candida krusei were incubated in porcine bile at AmB concentrations of 0.025-5.00 mg/L. In addition, patient bile samples retrieved for AmB quantification were inoculated with the same Candida strains. Biliary AmB concentrations were lower and displayed a slower rise and decline than plasma levels. The highest penetration ratio, as expressed by the ratio between the area under the AmB concentration-time curve in bile and plasma (liberated AmB) over the sampling period (AUC0-n bile/AUC0-n LI plasma), was 0.28. Proliferation of C. albicans and C. tropicalis in bile was similar to that in culture medium, whereas growth of C. glabrata was diminished and proliferation of C. krusei was absent in bile. In comparison with culture medium, AmB activity decreased in spiked porcine bile. In all but one patient bile sample, fungal growth was delayed or lacking even when AmB was not detectable. However, no fungicidal effect was observed in patient bile at AmB concentrations up to 1.28 mg/L. Thus, a reliable response of fungal cholangitis to treatment with L-AmB or ABCD cannot be anticipated.

Complement-Opsonized HIV-1 Overcomes Restriction in Dendritic Cells

PLoS Pathogens. Jun, 2015  |  Pubmed ID: 26121641

DCs express intrinsic cellular defense mechanisms to specifically inhibit HIV-1 replication. Thus, DCs are productively infected only at very low levels with HIV-1, and this non-permissiveness of DCs is suggested to go along with viral evasion. We now illustrate that complement-opsonized HIV-1 (HIV-C) efficiently bypasses SAMHD1 restriction and productively infects DCs including BDCA-1 DCs. Efficient DC infection by HIV-C was also observed using single-cycle HIV-C, and correlated with a remarkable elevated SAMHD1 T592 phosphorylation but not SAMHD1 degradation. If SAMHD1 phosphorylation was blocked using a CDK2-inhibitor HIV-C-induced DC infection was also significantly abrogated. Additionally, we found a higher maturation and co-stimulatory potential, aberrant type I interferon expression and signaling as well as a stronger induction of cellular immune responses in HIV-C-treated DCs. Collectively, our data highlight a novel protective mechanism mediated by complement opsonization of HIV to effectively promote DC immune functions, which might be in the future exploited to tackle HIV infection.

Treatment of Drug-resistant Aspergillus Infection

Expert Opinion on Pharmacotherapy. 2015  |  Pubmed ID: 26328502

Triazole antifungals are frontline drugs for the treatment and prophylaxis of infections due to Aspergillus species. Azole resistance is an emerging problem and is associated with treatment failure in several case series. The management of azole-resistant invasive aspergillosis remains a challenge and there are no guidelines with appropriate recommendations. The current clinical practice suggests that liposomal amphotericin B or a combination of voriconazole or posaconazole with an echinocandin may be effective. Although cross-resistance within the azoles seems to be common, the role of azoles in the management of azole-resistant aspergillosis remains unclear, but optimizing drug exposure is critical for treatment success.

Phylogenetic Diversity of Human Pathogenic Fusarium and Emergence of Uncommon Virulent Species

The Journal of Infection. Dec, 2015  |  Pubmed ID: 26348828

Fusarium species cause a broad spectrum of infections. However, little is known about the etiological agents to the species level. We identified Fusarium species isolated from clinical specimens including those of high risk patients to better understand the species involved in the pathogenesis.

A Point Prevalence Survey on Hand Hygiene, with a Special Focus on Candida Species

American Journal of Infection Control. Jan, 2016  |  Pubmed ID: 26320699

A 1-day point prevalence study evaluated hand hygiene compliance, yeast colonization, and contamination, focusing on the hands of health care workers (HCWs) and patient-oriented surfaces.

Natural Killer Cell-mediated Damage of Clinical Isolates of Mucormycetes

Mycoses. Jan, 2016  |  Pubmed ID: 26578394

Haematopoietic stem cell transplant (HSCT) recipients are at high risk for mucormycosis, which has a mortality of up to 90%. The adoptive transfer of Natural killer (NK) cells is a promising therapeutic option in order to improve the reconstitution of host immunity after HSCT and to directly combat the fungal pathogen. As a number of fungal pathogens have developed strategies to evade the innate immune system, we investigated the interaction of human NK cells with various clinical isolates of different species of mucormycetes. Our results show that human IL-2 prestimulated NK cells damaged all mucormycetes tested. The extent of the damage depended, at least in part, on the growth curve characteristics of the individual fungal isolate. All isolates decreased the secretion of interferon-γ by NK cells to a similar extent. Our data suggest that NK cells damage a wide spectrum of mucormycetes, but that the antifungal effect is higher if NK cells are administered at an early time point of infection.

Galleria Mellonella: An Invertebrate Model to Study Pathogenicity in Correctly Defined Fungal Species

Fungal Biology. Feb, 2016  |  Pubmed ID: 26781383

The high mortality rates and economic burden associated with fungal infections, plus the emergence of fungal strains resistant to antifungal drugs, make it necessary to get a deeper understanding of fungal pathogenesis, as well as to identify new target structures for antifungal drug development. Still, murine models are considered as the gold standard for studying pathogenesis, quantifying virulence, and analysing the efficacy of antifungal drugs. However, invertebrates, such as the larvae of the greater wax moth Galleria mellonella, are promising alternative hosts to address some of these questions, especially when a large number of fungal strains need to be evaluated. The purpose of this review is to summarize the benefits and drawbacks, explain the utilization of the invertebrate model host G. mellonella, and compare the virulence potential of the most important human fungal pathogens, with the focus on different virulence potential of closely related species.

Invasive Pulmonary Aspergillosis in Organ Transplants--Focus on Lung Transplants

Respiratory Investigation. Mar, 2016  |  Pubmed ID: 26879476

Infections with filamentous fungi are common in transplant recipients. The risk for aspergillosis and other invasive pulmonary mycosis (IPM) is high in patients undergoing stem cell and lung transplantations. The mortality rates range from 20% to 60% and depend on a number of risk factors. The typical manifestations of IPM are lung infiltrates, consolidations, and fungal tracheobronchitis. The most common infectious agent is Aspergillus fumigatus. Infections caused by non-Aspergillus molds are more frequent for various reasons. The species distribution of non-Aspergillus molds varies in different locations. Furthermore, infections caused by Mucor and Penicillium are increasing, as are infections caused by species resistant to azoles and amphotericin B. Most centers use antifungal prophylaxis with inhaled amphotericin B or oral azoles. Early diagnosis and therapy is crucial. Reliable information on the local microbiological spectrum is a prerequisite for the effective treatment of molds with primary or secondary resistance to antimycotic drugs.

IL-1 Receptor Antagonist Ameliorates Inflammasome-dependent Inflammation in Murine and Human Cystic Fibrosis

Nature Communications. Mar, 2016  |  Pubmed ID: 26972847

Dysregulated inflammasome activation contributes to respiratory infections and pathologic airway inflammation. Through basic and translational approaches involving murine models and human genetic epidemiology, we show here the importance of the different inflammasomes in regulating inflammatory responses in mice and humans with cystic fibrosis (CF), a life-threatening disorder of the lungs and digestive system. While both contributing to pathogen clearance, NLRP3 more than NLRC4 contributes to deleterious inflammatory responses in CF and correlates with defective NLRC4-dependent IL-1Ra production. Disease susceptibility in mice and microbial colonization in humans occurs in conditions of genetic deficiency of NLRC4 or IL-1Ra and can be rescued by administration of the recombinant IL-1Ra, anakinra. These results indicate that pathogenic NLRP3 activity in CF could be negatively regulated by IL-1Ra and provide a proof-of-concept evidence that inflammasomes are potential targets to limit the pathological consequences of microbial colonization in CF.

Lichtheimia Infection in a Lymphoma Patient: Case Report and a Brief Review of the Available Diagnostic Tools

Mycopathologia. Aug, 2016  |  Pubmed ID: 27115610

We describe the case of a patient with a T-lymphoblastic lymphoma whose disseminated mucormycosis was diagnosed with delay, and we address the diagnostic and therapeutic decision-making process and review the diagnostic workup of patients with potential IFD. The diagnosis was delayed despite a suggestive radiological presentation of the patient's pulmonary lesion. The uncommon risk profile (T-lymphoblastic lymphoma, short neutropenic phases) wrongly led to a low level of suspicion. The diagnosis was also hampered by the lack of indirect markers for infections caused by Mucorales, the low sensitivity of both fungal culture and panfungal PCR, and the limited availability of species-specific PCR. A high level of suspicion of IFD is needed, and aggressive diagnostic procedures should be promptly initiated even in apparently low-risk patients with uncommon presentations. The extent of the analytical workup should be decided on a case-by-case base. Diagnostic tests such as the galactomannan and β-D-glucan test and/or PCR on biological material followed by sequencing should be chosen according to their availability and after evaluation of their specificity and sensitivity. In high-risk patients, preemptive therapy with a broad-spectrum mould-active antifungal agent should be started before definitive diagnostic findings become available.

Long Term Complications Following 54 Consecutive Lung Transplants

Journal of Thoracic Disease. Jun, 2016  |  Pubmed ID: 27293842

Due to the complex therapy and the required high level of immunosuppression, lung recipients are at high risk to develop many different long term complications.

In Vitro Resistance of Clinical Fusarium Species to Amphotericin B and Voriconazole Using the EUCAST Antifungal Susceptibility Method

Diagnostic Microbiology and Infectious Disease. Aug, 2016  |  Pubmed ID: 27312690

Susceptibility testing using the EUCAST-AFST method against 39 clinical Fusarium strains consecutively collected from local and invasive infections during the last 10years assessed the in vitro activities of amphotericin B (AmB) and triazole antifungal agents. In addition, the susceptibility pattern of 12 reference strains from the CBS-KNAW Fungal Biodiversity Centre (CBS) was evaluated. In particular Fusarium petroliphilum and F. solani sensu lato were involved in disseminated infections and known for treatment failure. AmB displayed the lowest MICs followed by voriconazole VRC, posaconazole (POC). Itraconazole (ITC) showed high MIC values, displaying in vitro resistance. Clinical isolates were significantly (P <0.05) more resistant to AmB, VRC, and POC, than the CBS reference isolates probably due to previous exposure to antifungal therapy. Resistant profiles to AmB and VRC, which are the currently recommended agents in the guidelines for treatments, and a late diagnosis may be associated with high mortality rate in immunocompromised patients. The present antifungal susceptibility profiles showed that species- and strain-specific differences in antifungal susceptibility exist within Fusarium and that susceptibility testing is important and may improve the prognosis of these infections.

Granulomatous Encephalitis: Protothecosis Excluded?

Histopathology. Dec, 2016  |  Pubmed ID: 27334617

Common Genetic Polymorphisms Within NFκB-Related Genes and the Risk of Developing Invasive Aspergillosis

Frontiers in Microbiology. 2016  |  Pubmed ID: 27570521

Invasive Aspergillosis (IA) is an opportunistic infection caused by Aspergillus, a ubiquitously present airborne pathogenic mold. A growing number of studies suggest a major host genetic component in disease susceptibility. Here, we evaluated whether 14 single-nucleotide polymorphisms within NFκB1, NFκB2, RelA, RelB, Rel, and IRF4 genes influence the risk of IA in a population of 834 high-risk patients (157 IA and 677 non-IA) recruited through a collaborative effort involving the aspBIOmics consortium and four European clinical institutions. No significant overall associations between selected SNPs and the risk of IA were found in this large cohort. Although a hematopoietic stem cell transplantation (HSCT)-stratified analysis revealed that carriers of the IRF4 rs12203592T/T genotype had a six-fold increased risk of developing the infection when compared with those carrying the C allele (ORREC = 6.24, 95%CI 1.25-31.2, P = 0.026), the association of this variant with IA risk did not reach significance at experiment-wide significant threshold. In addition, we found an association of the IRF4AATC and IRF4GGTC haplotypes (not including the IRF4 rs12203592T risk allele) with a decreased risk of IA but the magnitude of the association was similar to the one observed in the single-SNP analysis, which indicated that the haplotypic effect on IA risk was likely due to the IRF4 rs12203592 SNP. Finally, no evidence of significant interactions among the genetic markers tested and the risk of IA was found. These results suggest that the SNPs on the studied genes do not have a clinically relevant impact on the risk of developing IA.

Therapeutische Umschau. Revue Therapeutique. 2016  |  Pubmed ID: 27731791

Aspergillus Fumigatus Responds to Natural Killer (NK) Cells with Upregulation of Stress Related Genes and Inhibits the Immunoregulatory Function of NK Cells

Oncotarget. Oct, 2016  |  Pubmed ID: 27738337

Natural Killer (NK) cells are active against Aspergillus fumigatus, which in turn is able to impair the host defense. Unfortunately, little is known on the mutual interaction of NK cells and A. fumigatus. We coincubated human NK cells with A. fumigatus hyphae and assessed the gene expression and protein concentration of selected molecules. We found that A. fumigatus up-regulates the gene expression of pro-inflammatory molecules in NK cells, but inhibited the release of these molecules resulting in intracellular accumulation and limited extracellular availability. A. fumigatus down-regulatedmRNA levels of perforin in NK cells, but increased its intra- and extracellular protein concentration. The gene expression of stress related molecules of A. fumigatus such as heat shock protein hsp90 was up-regulated by human NK cells. Our data characterize for the first time the immunosuppressive effect of A. fumigatus on NK cells and may help to develop new therapeutic antifungal strategies.

Clinical Evaluation of a Mucorales-specific Real-time PCR Assay in Tissue and Serum Samples

Journal of Medical Microbiology. Dec, 2016  |  Pubmed ID: 27902424

Molecular diagnostic assays can accelerate the diagnosis of fungal infections and subsequently improve patient outcomes. In particular, the detection of infections due to Mucorales is still challenging for laboratories and physicians. The aim of this study was to evaluate a probe-based Mucorales-specific real-time PCR assay (Muc18S) using tissue and serum samples from patients suffering from invasive mucormycosis (IMM). This assay can detect a broad range of clinically relevant Mucorales species and can be used to complement existing diagnostic tests or to screen high-risk patients. An advantage of the Muc18S assay is that it exclusively detects Mucorales species allowing the diagnosis of Mucorales DNA without sequencing within a few hours. In paraffin-embedded tissue samples this PCR-based method allowed rapid identification of Mucorales in comparison with standard methods and showed 91 % sensitivity in the IMM tissue samples. We also evaluated serum samples, an easily accessible material, from patients at risk from IMM. Mucorales DNA was detected in all patients with probable/proven IMM (100 %) and in 29 % of the possible cases. Detection of IMM in serum could enable an earlier diagnosis (up to 21 days) than current methods including tissue samples, which were gained mainly post-mortem. A screening strategy for high-risk patients, which would enable targeted treatment to improve patient outcomes, is therefore possible.

Nosocomial Outbreak of Extensively Drug-resistant Pseudomonas Aeruginosa Associated with Aromatherapy

American Journal of Infection Control. Nov, 2016  |  Pubmed ID: 27914649

An increase of extensively drug-resistant Pseudomonas aeruginosa (XDR-PA) in various clinical specimens among intensive care unit patients (n = 7) initiated an outbreak investigation consisting of patient data analyses, control of adherence to infection control guidelines, microbiologic surveys, and molecular-based studies. XDR-PA was detected in a jointly used aroma-oil nursing bottle for aromatherapy. We implemented the restriction of oil sharing among patients. Hence, the outbreak was controlled successfully.

Current Challenges in the Diagnosis of Fungal Infections

Methods in Molecular Biology (Clifton, N.J.). 2017  |  Pubmed ID: 27837496

Diagnosing fungal infections is a challenge, particularly in the immunocompromised host. Signs and symptoms are nonspecific, colonization is difficult to distinguish from invasive disease, blood cultures are commonly negative, and patients are often unable to undergo invasive diagnostic procedures. Culture and microscopic examination remain the "gold standard" but are insensitive. Antigen assays such as the galactomannan and glucan detection systems are frequently used, yet these tests vary in sensitivity and specificity, depending on the patient population involved. Molecular-based assays are not yet clinically validated.

Commercial Molecular Tests for Fungal Diagnosis from a Practical Point of View

Methods in Molecular Biology (Clifton, N.J.). 2017  |  Pubmed ID: 27837499

The increasing interest in molecular diagnostics is a result of tremendously improved knowledge on fungal infections in the past 20 years and the rapid development of new methods, in particular polymerase chain reaction. High expectations have been placed on molecular diagnostics, and the number of laboratories now using the relevant technology is rapidly increasing-resulting in an obvious need for standardization and definition of laboratory organization. In the past 10 years, multiple new molecular tools were marketed for the detection of DNA, antibodies, cell wall components, or other antigens. In contrast to classical culture methods, molecular methods do not detect a viable organisms, but only molecules which indicate its presence; this can be nucleic acids, cell components (antigens), or antibodies (Fig. 1). In this chapter, an overview is provided on commercially available detection tools, their strength and how to use them. A main focus is laid on providing tips and tricks that make daily life easier. We try to focus and mention methodical details which are not highlighted in the manufacturer's instructions of these test kits, but are based on our personal experience in the laboratory. Important to keep in mind is that molecular tools cannot replace culture, microscopy, or a critical view on patients' clinical history, signs, and symptoms, but provide a valuable add on tool. Diagnosis should not be based solely on a molecular test, but molecular tools might deliver an important piece of information that helps matching the diagnostic puzzle to a diagnosis, in particular as few tests are in vitro diagnostic tests (IVD) or only part of the whole test carries the IVD certificate (e.g., DNA extraction is often not included). Please be aware that the authors do not claim to provide a complete overview on all commercially available diagnostic assays being currently marketed for fungal detection, as those are subject to constant change. A main focus is put on commonly used panfungal assays and pathogen-specific assays, including Aspergillus-specific, Candida-specific, Cryptococcus specific, Histoplasma-specific, and Pneumocystis-specific assays. Assays are categorized according to their underlying principle in either antigen-detecting or antibody-detecting or DNA-detecting (Fig. 1). Other non-DNA-detecting nucleic acid methods such as FISH and PNA FISH are not summarized in this chapter and an overview on test performance, common false positives, and the clinical evaluation of commercial tests in studies is provided already in a previous book series by Javier Yugueros Marcos and David H. Pincus (Marcos and Pincus, Methods Mol Biol 968:25-54, 2013).

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