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1.  Positions and Numbers of FKS Mutations in Candida albicans Selectively Influence In Vitro and In Vivo Susceptibilities to Echinocandin Treatment 
Candidemia is the fourth most common kind of microbial bloodstream infection, with Candida albicans being the most common causative species. Echinocandins are employed as the first-line treatment for invasive candidiasis until the fungal species is determined and confirmed by clinical diagnosis. Echinocandins block the FKS glucan synthases responsible for embedding β-(1,3)-d-glucan in the cell wall. The increasing use of these drugs has led to the emergence of antifungal resistance, and elevated MICs have been associated with single-residue substitutions in specific hot spot regions of FKS1 and FKS2. Here, we show for the first time the caspofungin-mediated in vivo selection of a double mutation within one allele of the FKS1 hot spot 1 in a clinical isolate. We created a set of isogenic mutants and used a hematogenous murine model to evaluate the in vivo outcomes of echinocandin treatment. Heterozygous and homozygous double mutations significantly enhance the in vivo resistance of C. albicans compared with the resistance seen with heterozygous single mutations. The various FKS1 hot spot mutations differ in the degree of their MIC increase, substance-dependent in vivo response, and impact on virulence. Our results demonstrate that echinocandin EUCAST breakpoint definitions correlate with the in vivo response when a standard dosing regimen is used but cannot predict the in vivo response after a dose escalation. Moreover, patients colonized by a C. albicans strain with multiple mutations in FKS1 have a higher risk for therapeutic failure.
doi:10.1128/AAC.00123-14
PMCID: PMC4068606  PMID: 24733467
2.  Multilaboratory Study of Epidemiological Cutoff Values for Detection of Resistance in Eight Candida Species to Fluconazole, Posaconazole, and Voriconazole 
Although epidemiological cutoff values (ECVs) have been established for Candida spp. and the triazoles, they are based on MIC data from a single laboratory. We have established ECVs for eight Candida species and fluconazole, posaconazole, and voriconazole based on wild-type (WT) MIC distributions for isolates of C. albicans (n = 11,241 isolates), C. glabrata (7,538), C. parapsilosis (6,023), C. tropicalis (3,748), C. krusei (1,073), C. lusitaniae (574), C. guilliermondii (373), and C. dubliniensis (162). The 24-h CLSI broth microdilution MICs were collated from multiple laboratories (in Canada, Brazil, Europe, Mexico, Peru, and the United States). The ECVs for distributions originating from ≥6 laboratories, which included ≥95% of the modeled WT population, for fluconazole, posaconazole, and voriconazole were, respectively, 0.5, 0.06 and 0.03 μg/ml for C. albicans, 0.5, 0.25, and 0.03 μg/ml for C. dubliniensis, 8, 1, and 0.25 μg/ml for C. glabrata, 8, 0.5, and 0.12 μg/ml for C. guilliermondii, 32, 0.5, and 0.25 μg/ml for C. krusei, 1, 0.06, and 0.06 μg/ml for C. lusitaniae, 1, 0.25, and 0.03 μg/ml for C. parapsilosis, and 1, 0.12, and 0.06 μg/ml for C. tropicalis. The low number of MICs (<100) for other less prevalent species (C. famata, C. kefyr, C. orthopsilosis, C. rugosa) precluded ECV definition, but their MIC distributions are documented. Evaluation of our ECVs for some species/agent combinations using published individual MICs for 136 isolates (harboring mutations in or upregulation of ERG11, MDR1, CDR1, or CDR2) and 64 WT isolates indicated that our ECVs may be useful in distinguishing WT from non-WT isolates.
doi:10.1128/AAC.02615-13
PMCID: PMC4023759  PMID: 24419346
3.  Phylogenetic Relationships Matter: Antifungal Susceptibility among Clinically Relevant Yeasts 
The objective of this study was 2-fold: to evaluate whether phylogenetically closely related yeasts share common antifungal susceptibility profiles (ASPs) and whether these ASPs can be predicted from phylogeny. To address this question, 9,627 yeast strains were collected and tested for their antifungal susceptibility. Isolates were reidentified by considering recent changes in taxonomy and nomenclature. A phylogenetic (PHYLO) code based on the results of multilocus sequence analyses (large-subunit rRNA, small-subunit rRNA, translation elongation factor 1α, RNA polymerase II subunits 1 and 2) and the classification of the cellular neutral sugar composition of coenzyme Q and 18S ribosomal DNA was created to group related yeasts into PHYLO groups. The ASPs were determined for fluconazole, itraconazole, and voriconazole in each PHYLO group. The majority (95%) of the yeast strains were Ascomycetes. After reclassification, a total of 23 genera and 54 species were identified, resulting in an increase of 64% of genera and a decrease of 5% of species compared with the initial identification. These taxa were assigned to 17 distinct PHYLO groups (Ascomycota, n = 13; Basidiomycota, n = 4). ASPs for azoles were similar among members of the same PHYLO group and different between the various PHYLO groups. Yeast phylogeny may be an additional tool to significantly enhance the assessment of MIC values and to predict antifungal susceptibility, thereby more rapidly initiating appropriate patient management.
doi:10.1128/AAC.01799-13
PMCID: PMC3957855  PMID: 24366735
4.  Multicenter Study of Anidulafungin and Micafungin MIC Distributions and Epidemiological Cutoff Values for Eight Candida Species and the CLSI M27-A3 Broth Microdilution Method 
Since epidemiological cutoff values (ECVs) using CLSI MICs from multiple laboratories are not available for Candida spp. and the echinocandins, we established ECVs for anidulafungin and micafungin on the basis of wild-type (WT) MIC distributions (for organisms in a species-drug combination with no detectable acquired resistance mechanisms) for 8,210 Candida albicans, 3,102 C. glabrata, 3,976 C. parapsilosis, 2,042 C. tropicalis, 617 C. krusei, 258 C. lusitaniae, 234 C. guilliermondii, and 131 C. dubliniensis isolates. CLSI broth microdilution MIC data gathered from 15 different laboratories in Canada, Europe, Mexico, Peru, and the United States were aggregated to statistically define ECVs. ECVs encompassing 97.5% of the statistically modeled population for anidulafungin and micafungin were, respectively, 0.12 and 0.03 μg/ml for C. albicans, 0.12 and 0.03 μg/ml for C. glabrata, 8 and 4 μg/ml for C. parapsilosis, 0.12 and 0.06 μg/ml for C. tropicalis, 0.25 and 0.25 μg/ml for C. krusei, 1 and 0.5 μg/ml for C. lusitaniae, 8 and 2 μg/ml for C. guilliermondii, and 0.12 and 0.12 μg/ml for C. dubliniensis. Previously reported single and multicenter ECVs defined in the present study were quite similar or within 1 2-fold dilution of each other. For a collection of 230 WT isolates (no fks mutations) and 51 isolates with fks mutations, the species-specific ECVs for anidulafungin and micafungin correctly classified 47 (92.2%) and 51 (100%) of the fks mutants, respectively, as non-WT strains. These ECVs may aid in detecting non-WT isolates with reduced susceptibility to anidulafungin and micafungin due to fks mutations.
doi:10.1128/AAC.02020-13
PMCID: PMC3910887  PMID: 24277027
5.  Interlaboratory Variability of Caspofungin MICs for Candida spp. Using CLSI and EUCAST Methods: Should the Clinical Laboratory Be Testing This Agent? 
Antimicrobial Agents and Chemotherapy  2013;57(12):5836-5842.
Although Clinical and Laboratory Standards Institute (CLSI) clinical breakpoints (CBPs) are available for interpreting echinocandin MICs for Candida spp., epidemiologic cutoff values (ECVs) based on collective MIC data from multiple laboratories have not been defined. While collating CLSI caspofungin MICs for 145 to 11,550 Candida isolates from 17 laboratories (Brazil, Canada, Europe, Mexico, Peru, and the United States), we observed an extraordinary amount of modal variability (wide ranges) among laboratories as well as truncated and bimodal MIC distributions. The species-specific modes across different laboratories ranged from 0.016 to 0.5 μg/ml for C. albicans and C. tropicalis, 0.031 to 0.5 μg/ml for C. glabrata, and 0.063 to 1 μg/ml for C. krusei. Variability was also similar among MIC distributions for C. dubliniensis and C. lusitaniae. The exceptions were C. parapsilosis and C. guilliermondii MIC distributions, where most modes were within one 2-fold dilution of each other. These findings were consistent with available data from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (403 to 2,556 MICs) for C. albicans, C. glabrata, C. krusei, and C. tropicalis. Although many factors (caspofungin powder source, stock solution solvent, powder storage time length and temperature, and MIC determination testing parameters) were examined as a potential cause of such unprecedented variability, a single specific cause was not identified. Therefore, it seems highly likely that the use of the CLSI species-specific caspofungin CBPs could lead to reporting an excessive number of wild-type (WT) isolates (e.g., C. glabrata and C. krusei) as either non-WT or resistant isolates. Until this problem is resolved, routine testing or reporting of CLSI caspofungin MICs for Candida is not recommended; micafungin or anidulafungin data could be used instead.
doi:10.1128/AAC.01519-13
PMCID: PMC3837874  PMID: 24018263
6.  Multicenter Study of Isavuconazole MIC Distributions and Epidemiological Cutoff Values for Aspergillus spp. for the CLSI M38-A2 Broth Microdilution Method 
Epidemiological cutoff values (ECVs) were established for the new triazole isavuconazole and Aspergillus species wild-type (WT) MIC distributions (organisms in a species-drug combination with no detectable acquired resistance mechanisms) that were defined with 855 Aspergillus fumigatus, 444 A. flavus, 106 A. nidulans, 207 A. niger, 384 A. terreus, and 75 A. versicolor species complex isolates; 22 Aspergillus section Usti isolates were also included. CLSI broth microdilution MIC data gathered in Europe, India, Mexico, and the United States were aggregated to statistically define ECVs. ECVs were 1 μg/ml for the A. fumigatus species complex, 1 μg/ml for the A. flavus species complex, 0.25 μg/ml for the A. nidulans species complex, 4 μg/ml for the A. niger species complex, 1 μg/ml for the A. terreus species complex, and 1 μg/ml for the A. versicolor species complex; due to the small number of isolates, an ECV was not proposed for Aspergillus section Usti. These ECVs may aid in detecting non-WT isolates with reduced susceptibility to isavuconazole due to cyp51A (an A. fumigatus species complex resistance mechanism among the triazoles) or other mutations.
doi:10.1128/AAC.00636-13
PMCID: PMC3719700  PMID: 23716059
7.  Cryptococcus neoformans-Cryptococcus gattii Species Complex: an International Study of Wild-Type Susceptibility Endpoint Distributions and Epidemiological Cutoff Values for Fluconazole, Itraconazole, Posaconazole, and Voriconazole 
Antimicrobial Agents and Chemotherapy  2012;56(11):5898-5906.
Epidemiological cutoff values (ECVs) for the Cryptococcus neoformans-Cryptococcus gattii species complex versus fluconazole, itraconazole, posaconazole, and voriconazole are not available. We established ECVs for these species and agents based on wild-type (WT) MIC distributions. A total of 2,985 to 5,733 CLSI MICs for C. neoformans (including isolates of molecular type VNI [MICs for 759 to 1,137 isolates] and VNII, VNIII, and VNIV [MICs for 24 to 57 isolates]) and 705 to 975 MICs for C. gattii (including 42 to 260 for VGI, VGII, VGIII, and VGIV isolates) were gathered in 15 to 24 laboratories (Europe, United States, Argentina, Australia, Brazil, Canada, Cuba, India, Mexico, and South Africa) and were aggregated for analysis. Additionally, 220 to 359 MICs measured using CLSI yeast nitrogen base (YNB) medium instead of CLSI RPMI medium for C. neoformans were evaluated. CLSI RPMI medium ECVs for distributions originating from at least three laboratories, which included ≥95% of the modeled WT population, were as follows: fluconazole, 8 μg/ml (VNI, C. gattii nontyped, VGI, VGIIa, and VGIII), 16 μg/ml (C. neoformans nontyped, VNIII, and VGIV), and 32 μg/ml (VGII); itraconazole, 0.25 μg/ml (VNI), 0.5 μg/ml (C. neoformans and C. gattii nontyped and VGI to VGIII), and 1 μg/ml (VGIV); posaconazole, 0.25 μg/ml (C. neoformans nontyped and VNI) and 0.5 μg/ml (C. gattii nontyped and VGI); and voriconazole, 0.12 μg/ml (VNIV), 0.25 μg/ml (C. neoformans and C. gattii nontyped, VNI, VNIII, VGII, and VGIIa,), and 0.5 μg/ml (VGI). The number of laboratories contributing data for other molecular types was too low to ascertain that the differences were due to factors other than assay variation. In the absence of clinical breakpoints, our ECVs may aid in the detection of isolates with acquired resistance mechanisms and should be listed in the revised CLSI M27-A3 and CLSI M27-S3 documents.
doi:10.1128/AAC.01115-12
PMCID: PMC3486550  PMID: 22948877
8.  Improvement of Detection of Bacterial Pathogens in Normally Sterile Body Sites with a Focus on Orthopedic Samples by Use of a Commercial 16S rRNA Broad-Range PCR and Sequence Analysis 
Journal of Clinical Microbiology  2012;50(7):2250-2254.
A new commercially available universal 16S and 18S rRNA gene PCR test, which is followed by sequence analysis of amplicons (SepsiTest), was evaluated for rapid identification of pathogens in the diagnosis of bone and joint infections. Eighty-three orthopedic samples and 21 specimens from other normally sterile body sites collected from 84 patients were analyzed in parallel by culture and PCR for detection of bacteria and fungi. Compared to culture, the diagnostic sensitivity and specificity of PCR were 88.5% and 83.5%, respectively. The detection rate of PCR (34.6%) was higher than that of bacterial culture (25.0%) as a consequence of the presence of fastidious and noncultivable species in samples and antibiotic treatment of patients. Thirteen culture-negative infections were identified by PCR, and PCR was able to detect culture-proven polymicrobial infections. On the other hand, three samples were culture positive but PCR negative. SepsiTest was demonstrated to be a valuable supplemental tool in the rapid detection of bacteria, especially for fastidious and noncultivable organisms, allowing earlier initiation of pathogen-adapted therapy in patients with bone and joint infections.
doi:10.1128/JCM.00362-12
PMCID: PMC3405601  PMID: 22553237
9.  Cryptococcus neoformans-Cryptococcus gattii Species Complex: an International Study of Wild-Type Susceptibility Endpoint Distributions and Epidemiological Cutoff Values for Amphotericin B and Flucytosine 
Clinical breakpoints (CBPs) are not available for the Cryptococcus neoformans-Cryptococcus gattii species complex. MIC distributions were constructed for the wild type (WT) to establish epidemiologic cutoff values (ECVs) for C. neoformans and C. gattii versus amphotericin B and flucytosine. A total of 3,590 amphotericin B and 3,045 flucytosine CLSI MICs for C. neoformans (including 1,002 VNI isolates and 8 to 39 VNII, VNIII, and VNIV isolates) and 985 and 853 MICs for C. gattii, respectively (including 42 to 259 VGI, VGII, VGIII, and VGIV isolates), were gathered in 9 to 16 (amphotericin B) and 8 to 13 (flucytosine) laboratories (Europe, United States, Australia, Brazil, Canada, India, and South Africa) and aggregated for the analyses. Additionally, 442 amphotericin B and 313 flucytosine MICs measured by using CLSI-YNB medium instead of CLSI-RPMI medium and 237 Etest amphotericin B MICs for C. neoformans were evaluated. CLSI-RPMI ECVs for distributions originating in ≥3 laboratories (with the percentages of isolates for which MICs were less than or equal to ECVs given in parentheses) were as follows: for amphotericin B, 0.5 μg/ml for C. neoformans VNI (97.2%) and C. gattii VGI and VGIIa (99.2 and 97.5%, respectively) and 1 μg/ml for C. neoformans (98.5%) and C. gattii nontyped (100%) and VGII (99.2%) isolates; for flucytosine, 4 μg/ml for C. gattii nontyped (96.4%) and VGI (95.7%) isolates, 8 μg/ml for VNI (96.6%) isolates, and 16 μg/ml for C. neoformans nontyped (98.6%) and C. gattii VGII (97.1%) isolates. Other molecular types had apparent variations in MIC distributions, but the number of laboratories contributing data was too low to allow us to ascertain that the differences were due to factors other than assay variation. ECVs may aid in the detection of isolates with acquired resistance mechanisms.
doi:10.1128/AAC.06252-11
PMCID: PMC3370763  PMID: 22391546
10.  Epidemiology and antifungal resistance in invasive aspergillosis according to primary disease - review of the literature 
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.
doi:10.1186/2047-783X-16-4-153
PMCID: PMC3352071  PMID: 21486729
Epidemiology; antifungal resistance; Aspergillus ; azoles; candins
11.  Interaction of serotonin with Candida albicans selectively attenuates fungal virulence in vitro 
In this study we investigated whether the direct interaction between Candida albicans CBS 5982 and 5-hydroxytryptamine (5-HT) alters candidial virulence. Hyphae elongation, phospholipase activity and the production of secreted aspartyl proteinases (Saps) following 5-HT treatment were investigated. 5-HT treatment of C. albicans significantly (P < 0.05) affected hyphal extension, phospholipase activity and the production of Saps at concentrations of 118–0.46 mM. In conclusion, our findings suggest that the interaction between 5-HT and C. albicans may diminish the virulence properties of this fungal pathogen.
doi:10.1016/j.ijantimicag.2005.07.006
PMCID: PMC2980867  PMID: 16157477
Candida albicans; 5-Hydroxytryptamine (5-HT); Virulence factor; Antifungal activity
12.  PCR based identification and discrimination of agents of mucormycosis and aspergillosis in paraffin wax embedded tissue 
Journal of Clinical Pathology  2005;58(11):1180-1184.
Background: Invasive fungal infections are often diagnosed by histopathology without identification of the causative fungi, which show significantly different antifungal susceptibilities.
Aims: To establish and evaluate a system of two seminested polymerase chain reaction (PCR) assays to identify and discriminate between agents of aspergillosis and mucormycosis in paraffin wax embedded tissue samples.
Methods: DNA of 52 blinded samples from five different centres was extracted and used as a template in two PCR assays targeting the mitochondrial aspergillosis DNA and the 18S ribosomal DNA of zygomycetes.
Results: Specific fungal DNA was identified in 27 of 44 samples in accordance with a histopathological diagnosis of zygomycosis or aspergillosis, respectively. Aspergillus fumigatus DNA was amplified from one specimen of zygomycosis (diagnosed by histopathology). In four of 16 PCR negative samples no human DNA was amplified, possibly as a result of the destruction of DNA before paraffin wax embedding. In addition, eight samples from clinically suspected fungal infections (without histopathological proof) were examined. The two PCR assays detected a concomitant infection with Absidia corymbifera and A fumigatus in one, and infections with Rhizopus arrhizus and A fumigatus in another two cases.
Conclusions: The two seminested PCR assays described here can support a histopathological diagnosis of mucormycosis or aspergillosis, and can identify the infective agent, thereby optimising antifungal treatment.
doi:10.1136/jcp.2004.024703
PMCID: PMC1770765  PMID: 16254108
mucormycosis; aspergillosis; zygomycetes; polymerase chain reaction

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