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1.  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 
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.
PMCID: PMC4068555  PMID: 24733474
2.  De Novo Whole-Genome Sequence and Genome Annotation of Lichtheimia ramosa 
Genome Announcements  2014;2(5):e00888-14.
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.
PMCID: PMC4161746  PMID: 25212617
3.  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;9(7):e103113.
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.
PMCID: PMC4114486  PMID: 25072733
4.  Determination of Isavuconazole Susceptibility of Aspergillus and Candida Species by the EUCAST Method 
Antimicrobial Agents and Chemotherapy  2013;57(11):5426-5431.
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.
PMCID: PMC3811258  PMID: 23959309
5.  New Insight into Amphotericin B Resistance in Aspergillus terreus 
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.
PMCID: PMC3623369  PMID: 23318794
6.  Utility of PCR in Diagnosis of Invasive Fungal Infections: Real-Life Data from a Multicenter Study 
Journal of Clinical Microbiology  2013;51(3):863-868.
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.
PMCID: PMC3592065  PMID: 23269732
7.  Why is biopsy of suspected fungal lung lesions necessary?☆ 
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.
PMCID: PMC3885920  PMID: 24432240
Childhood; Invasive pulmonary infections; CT-guided biopsy; Aspergillus flavus; Amphothericin-B
8.  Interaction of Platelets and Anidulafungin against Aspergillus fumigatus 
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.
PMCID: PMC3535898  PMID: 23114752
9.  CHD1 Contributes to Intestinal Resistance against Infection by P. aeruginosa in Drosophila melanogaster 
PLoS ONE  2012;7(8):e43144.
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.
PMCID: PMC3418260  PMID: 22912810
10.  Preclinical evaluation of two 68Ga-siderophores as potential radiopharmaceuticals for Aspergillus fumigatus infection imaging 
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 68Ga and can be used for PET imaging of A. fumigatus infection in rats. Here we report on the further evaluation of the most promising 68Ga-siderophore candidates, triacetylfusarinine (TAFC) and ferrioxamine E (FOXE).
Siderophores were labelled with 68Ga using acetate buffer. Log P, protein binding and stability values were determined. Uptake by A. fumigatus was studied in vitro in cultures with high and low iron loads. In vivo biodistribution was determined in normal mice and an infection model was established using neutropenic rats inoculated with A. fumigatus. Static and dynamic μPET imaging was performed and correlated with CT images, and lung infection was evaluated ex vivo.
68Ga-siderophores were labelled with high radiochemical purity and specific activity. 68Ga-TAFC and 68Ga-FOXE showed high uptake by A. fumigatus in iron-deficient cultures. In normal mice, 68Ga-TAFC and 68Ga-FOXE showed rapid renal excretion with high metabolic stability. In the rat infection model focal lung uptake was detected by μPET with both compounds and increased with severity of the infection, correlating with abnormal CT images.
68Ga-TAFC and 68Ga-FOXE displayed excellent in vitro stability and high uptake by A. fumigatus. Both compounds showed excellent pharmacokinetics, highly selective accumulation in infected lung tissue and good correlation with severity of disease in a rat infection model, which makes them promising agents for A. fumigatus infection imaging.
Electronic supplementary material
The online version of this article (doi:10.1007/s00259-012-2110-3) contains supplementary material, which is available to authorized users.
PMCID: PMC3369139  PMID: 22526953
68Ga-siderophores; Aspergillus fumigatus; Invasive aspergillosis; Infection imaging
11.  Inhibition of Human Immunodeficiency Virus Replication by Cell Membrane–Crossing Oligomers 
Molecular Medicine  2011;18(1):111-122.
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.
PMCID: PMC3276398  PMID: 22105607
12.  Environmental Study of Azole-Resistant Aspergillus fumigatus and Other Aspergilli in Austria, Denmark, and Spain ▿  
Antimicrobial Agents and Chemotherapy  2010;54(11):4545-4549.
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.
PMCID: PMC2976122  PMID: 20805399
13.  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  2011;49(5):1872-1878.
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.
PMCID: PMC3122670  PMID: 21367988
14.  Potential Antifungal Effects of Human Platelets against Zygomycetes In Vitro 
The Journal of infectious diseases  2009;200(7):1176-1179.
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.
PMCID: PMC3017871  PMID: 19698079
15.  Comparison of Caspofungin MICs by Means of EUCAST Method EDef 7.1 Using Two Different Concentrations of Glucose▿  
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.
PMCID: PMC2897297  PMID: 20479199
16.  Epidemiology of Invasive Fungal Infections in the Mediterranean Area 
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.
PMCID: PMC3103242  PMID: 21625305
17.  Interaction of 5-hydroxytryptamine (serotonin) against Aspergillus spp. in vitro 
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 30 min at 4°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 4 h 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.
PMCID: PMC3010239  PMID: 17276041
Serotonin; Aspergillus spp.; Ergosterol; Platelets
18.  68Ga-Siderophores for PET Imaging of Invasive Pulmonary Aspergillosis: Proof of Principle 
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 68Ga, a positron emitter with complexing properties comparable to those of Fe(III), as a radiopharmaceutical for imaging IPA.
68Ga radiolabeling of the A. fumigatus siderophores desferri-triacetylfusarinine C (TAFC) and desferri-ferricrocin (FC) was performed at high specific activity. Stability, protein binding, and log P values were determined. In vitro uptake in A. fumigatus cultures was tested under varying conditions. Biodistribution was studied in healthy noninfected BALB/c mice, and uptake was studied in a model of A. fumigatus infection using immunosuppressed Lewis rats.
High-specific-activity 68Ga labeling could be achieved, and resulting complexes were stable in serum, toward diethylenetriaminepentaacetic acid and Fe(III) challenge. Both siderophores showed hydrophilic properties (68Ga-TAFC, log P = −2.59; 68Ga-FC, log P = −3.17) with low values of protein binding for 68Ga-TAFC (<2%). Uptake of both siderophores was highly dependent on the mycelial iron load and could be blocked with an excess (10 μM) of siderophore or NaN3, indicating specific, energy-dependent uptake. In noninfected mice, 68Ga-TAFC showed rapid renal excretion and low blood values (1.6 ± 0.37 percentage injected dose per gram [%ID/g] at 30 min); in urine only intact 68Ga-TAFC was detected. In contrast, 68Ga-FC revealed high retention in blood (16.1 ± 1.07 %ID/g at 90 min) and rapid metabolism. In the rat IPA model, lung uptake of 68Ga-TAFC was dependent on the severity of infection, with less than 0.04 %ID/g in control rats (n = 5) and 0.29 ± 0.11 %ID/g in mildly infected (n = 3) and 0.95 ± 0.37 %ID/g in severely infected (n = 4) rats. PET showed focal accumulation in infected lung tissue.
Both siderophores bound 68Ga with high affinity, and 68Ga-TAFC, especially, showed high stability. 68Ga-TAFC displayed highly selective accumulation by A. fumigatus subspecies in vitro and in vivo. The high and specific uptake by A. fumigatus proves the potential of 68Ga-labeled siderophores for the specific detection of A. fumigatus during infection. They hold promise as new PET agents for IPA.
PMCID: PMC2992174  PMID: 20351354
invasive pulmonary aspergillosis; fungal infection; siderophores; 68Ga; PET
19.  Human Platelets Attenuate Aspergillus Species via Granule-Dependent Mechanisms 
The Journal of infectious diseases  2008;198(8):1243-1246.
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 [3H]-serotonin (5-hydroxytryptamine [5-HT]) was performed. Exposure to platelets resulted in significantly decreased GM release (P<.05), hyphal elongation (P<.001), colony size, pig-mentation, 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.
PMCID: PMC2980866  PMID: 18752432
20.  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▿  
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 MIC50) 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 ≤ 2 μg/ml) for CLSI results, 89.2% fks hot spot mutants were classified as anidulafungin susceptible, 60.7% as caspofungin susceptible, and 92.9% as micafungin susceptible. In conclusion, no test was perfect, but anidulafungin susceptibility testing using the WT-UL to define susceptibility reliably identified fks hot spot mutants.
PMCID: PMC2798528  PMID: 19884370
22.  Aspergillus alabamensis, a New Clinically Relevant Species in the Section Terrei▿  
Eukaryotic Cell  2009;8(5):713-722.
Phylogenetic analyses of sequences generated from portions of three genes coding for the proteins enolase (enoA), β-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.
PMCID: PMC2681604  PMID: 19304950
23.  In Vitro Activity of Isavuconazole against Aspergillus Species and Zygomycetes According to the Methodology of the European Committee on Antimicrobial Susceptibility Testing▿  
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.
PMCID: PMC2663115  PMID: 19164153
24.  Breakthrough Aspergillus fumigatus and Candida albicans Double Infection during Caspofungin Treatment: Laboratory Characteristics and Implication for Susceptibility Testing▿  
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 μg/ml caspofungin and 0.5 μg/ml anidulafungin by Etest, 2 μg/ml caspofungin and 0.125 μg/ml anidulafungin by EUCAST methods, and 1 μg/ml caspofungin and 0.5 μg/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.
PMCID: PMC2650576  PMID: 19104024
25.  In Vitro Activities of Various Antifungal Drugs against Aspergillus terreus: Global Assessment Using the Methodology of the European Committee on Antimicrobial Susceptibility Testing▿  
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.
PMCID: PMC2630662  PMID: 19064891

Results 1-25 (42)