Etest ECVs/ECOFFs for Detection of Resistance in Prevalent and Three Nonprevalent Candida spp. to Triazoles and Amphotericin B and Aspergillus spp. to Caspofungin: Further Assessment of Modal Variability.

Susceptibility testing is an important tool in the clinical setting; its utility is based on the availability of categorical endpoints, breakpoints (BPs), or epidemiological cutoff values (ECVs/ECOFFs). CLSI and EUCAST have developed antifungal susceptibility testing, BPs, and ECVs for some fungal species. Although the concentration gradient strip bioMérieux Etest is useful for routine testing in the clinical laboratory, ECVs are not available for all agent/species; the lack of clinical data precludes development of BPs. We reevaluated and consolidated Etest data points from three previous studies and included new data. We defined ECOFFinder Etest ECVs for three sets of species-agent combinations: fluconazole, posaconazole, and voriconazole and 9 Candida spp.; amphotericin B and 3 nonprevalent Candida spp.; and caspofungin and 4 Aspergillus spp. The total of Etest MICs from 23 laboratories (Europe, the Americas, and South Africa) included (antifungal agent dependent): 17,242 Candida albicans, 244 C. dubliniensis, 5,129 C. glabrata species complex (SC), 275 C. guilliermondii (Meyerozyma guilliermondii), 1,133 C. krusei (Pichia kudriavzevii), 933 C. kefyr (Kluyveromyces marxianus), 519 C. lusitaniae (Clavispora lusitaniae), 2,947 C. parapsilosis SC, 2,214 C. tropicalis, 3,212 Aspergillus fumigatus, 232 A. flavus, 181 A. niger, and 267 A. terreus SC isolates. Triazole MICs for 66 confirmed non-wild-type (non-WT) Candida isolates were available (ERG11 point mutations). Distributions fulfilling CLSI ECV criteria were pooled, and ECOFFinder Etest ECVs were established for triazoles (9 Candida spp.), amphotericin B (3 less-prevalent Candida spp.), and caspofungin (4 Aspergillus spp.). Etest fluconazole ECVs could be good detectors of Candida non-WT isolates (59/61 non-WT, 4 of 6 species).

Interlaboratory evaluation of Mucorales PCR assays for testing serum specimens: A study by the fungal PCR Initiative and the Modimucor study group.

Interlaboratory evaluations of Mucorales qPCR assays were developed to assess the reproducibility and performance of methods currently used. The participants comprised 12 laboratories from French university hospitals (nine of them participating in the Modimucor study) and 11 laboratories participating in the Fungal PCR Initiative. For panel 1, three sera were each spiked with DNA from three different species (Rhizomucor pusillus, Lichtheimia corymbifera, Rhizopus oryzae). For panel 2, six sera with three concentrations of R. pusillus and L. corymbifera (1, 10, and 100 genomes/ml) were prepared. Each panel included a blind negative-control serum. A form was distributed with each panel to collect results and required technical information, including DNA extraction method, sample volume used, DNA elution volume, qPCR method, qPCR template input volume, qPCR total reaction volume, qPCR platform, and qPCR reagents used. For panel 1, assessing 18 different protocols, qualitative results (positive or negative) were correct in 97% of cases (70/72). A very low interlaboratory variability in Cq values (SD = 1.89 cycles) were observed. For panel 2 assessing 26 different protocols, the detection rates were high (77-100%) for 5/6 of spiked serum. There was a significant association between the qPCR platform and performance. However, certain technical steps and optimal combinations of factors may also impact performance. The good reproducibility and performance demonstrated in this study support the use of Mucorales qPCR as part of the diagnostic strategy for mucormycosis.

N-chlorotaurine, a potent weapon against multiresistant bacteria.

AIMS: N-chlorotaurine (NCT) is a body-own mild oxidizing antiseptic that can be applied topically as a well-tolerated anti-infective at many body sites. The objective of this study was to demonstrate its activity against representative nosocomial multidrug-resistant bacteria. METHODS AND RESULTS: The bactericidal activity of NCT was tested in quantitative killing assays against a panel of multiresistant Gram-positive and Gram-negative clinical isolates. N-chlorotaurine (1%, 55 mmol l-1 ) reduced the number of CFU of strains of methicillin-resistant Staphylococcus aureus, linezolid-resistant Staphylococcus epidermidis, vancomycin-resistant, and linezolid- and vancomycin-resistant Enterococcus faecium, 3MRGN and 4MRGN Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae by at least 2 log10 steps after 15 min and completely or nearly to the detection limit after 30 min at pH 7·1 and 37°C. CONCLUSION: The activity of NCT against these clinical isolates is similar to that against non-resistant ATCC strains and therefore not influenced by antibiotic resistance. This can be explained by the oxidizing and chlorinating mechanism of action of NCT, which leads to an attack of multiple targets in the microorganisms. SIGNIFICANCE AND IMPACT OF THE STUDY: The bactericidal spectrum of NCT is not restricted by resistance against antibiotics. Therefore, it can be used against resistant strains, too.

Polymorphisms within the ARNT2 and CX3CR1 Genes Are Associated with the Risk of Developing Invasive Aspergillosis.

Invasive aspergillosis (IA) is a life-threatening infection that affects an increasing number of patients undergoing chemotherapy or allo-transplantation, and recent studies have shown that genetic factors contribute to disease susceptibility. In this two-stage, population-based, case-control study, we evaluated whether 7 potentially functional single nucleotide polymorphisms (SNPs) within the ARNT2 and CX3CR1 genes influence the risk of IA in high-risk hematological patients. We genotyped selected SNPs in a cohort of 500 hematological patients (103 of those had been diagnosed with proven or probable IA), and we evaluated their association with the risk of developing IA. The association of the most interesting markers of IA risk was then validated in a replication population, including 474 subjects (94 IA and 380 non-IA patients). Functional experiments were also performed to confirm the biological relevance of the most interesting markers. The meta-analysis of both populations showed that carriers of the ARNT2rs1374213G, CX3CR1rs7631529A, and CX3CR1rs9823718G alleles (where the RefSeq identifier appears as a subscript) had a significantly increased risk of developing IA according to a log-additive model (P value from the meta-analysis [PMeta] = 9.8 · 10-5, PMeta = 1.5 · 10-4, and PMeta =7.9 · 10-5, respectively). Haplotype analysis also confirmed the association of the CX3CR1 haplotype with AG CGG with an increased risk of IA (P = 4.0 · 10-4). Mechanistically, we observed that monocyte-derived macrophages (MDM) from subjects carrying the ARNTR2rs1374213G allele or the GG genotype showed a significantly impaired fungicidal activity but that MDM from carriers of the ARNT2rs1374213G and CX3CR1rs9823718G or CX3CR1rs7631529A alleles had deregulated immune responses to Aspergillus conidia. These results, together with those from expression quantitative trait locus (eQTL) data browsers showing a strong correlation of the CX3CR1rs9823718G allele with lower levels of CX3CR1 mRNA in whole peripheral blood (P = 2.46 · 10-7) and primary monocytes (P = 4.31 · 10-7), highlight the role of the ARNT2 and CX3CR1 loci in modulating and predicting IA risk and provide new insights into the host immune mechanisms involved in IA development.

Candida in the Respiratory Tract Potentially Triggers Galactomannan Positivity in Nonhematological Patients.

BAL fluid samples from critically ill patients shared a rate of 29% false-positive galactomannan results. We aimed to determine whether Candida species abundance in BAL fluid causes galactomannan (GM) positivity. A total of 89 Candida culture-positive BAL fluid samples from patients without suspicion of invasive aspergillosis (IA) were analyzed. GM results were correlated with Candida species abundance, Candida species quantity, and patient data. Candida species quantities of ≥104/ml and Candida glabrata abundance were significantly associated with positive GM results. The added diagnostic value of GM in BAL fluid for diagnosing IA in critically ill patients is limited.

Method-Dependent Epidemiological Cutoff Values for Detection of Triazole Resistance in Candida and Aspergillus Species for the Sensititre YeastOne Colorimetric Broth and Etest Agar Diffusion Methods.

Although the Sensititre Yeast-One (SYO) and Etest methods are widely utilized, interpretive criteria are not available for triazole susceptibility testing of Candida or Aspergillus species. We collected fluconazole, itraconazole, posaconazole, and voriconazole SYO and Etest MICs from 39 laboratories representing all continents for (method/agent-dependent) 11,171 Candida albicans, 215 C. dubliniensis, 4,418 C. glabrata species complex, 157 C.guilliermondii (Meyerozyma guilliermondii), 676 C. krusei (Pichia kudriavzevii), 298 C.lusitaniae (Clavispora lusitaniae), 911 C.parapsilosissensu stricto, 3,691 C.parapsilosis species complex, 36 C.metapsilosis, 110 C.orthopsilosis, 1,854 C.tropicalis, 244 Saccharomyces cerevisiae, 1,409 Aspergillus fumigatus, 389 A.flavus, 130 A.nidulans, 233 A.niger, and 302 A.terreus complex isolates. SYO/Etest MICs for 282 confirmed non-wild-type (non-WT) isolates were included: ERG11 (C. albicans), ERG11 and MRR1 (C. parapsilosis), cyp51A (A. fumigatus), and CDR2 and CDR1 overexpression (C. albicans and C. glabrata, respectively). Interlaboratory modal agreement was superior by SYO for yeast species and by the Etest for Aspergillus spp. Distributions fulfilling CLSI criteria for epidemiological cutoff value (ECV) definition were pooled, and we proposed SYO ECVs for S. cerevisiae and 9 yeast and 3 Aspergillus species and Etest ECVs for 5 yeast and 4 Aspergillus species. The posaconazole SYO ECV of 0.06 µg/ml for C. albicans and the Etest itraconazole ECV of 2 µg/ml for A. fumigatus were the best predictors of non-WT isolates. These findings support the need for method-dependent ECVs, as, overall, the SYO appears to perform better for susceptibility testing of yeast species and the Etest appears to perform better for susceptibility testing of Aspergillus spp. Further evaluations should be conducted with more Candida mutants.

Direct detection of Exophiala and Scedosporium species in sputa of patients with cystic fibrosis.

Detection of species of Exophiala and Scedosporium in the respiratory tracts of cystic fibrosis (CF) patients remains controversial because of highly variable results. The results of our study suggested a significantly higher prevalence and more complex colonization than previously estimated. Approximately 17% (27/162) of clinical sputum samples were found to be positive for Exophiala dermatitidis and 30% (49/162) were positive for Scedosporium apiospermum / S. boydii species complex determined by reverse line blot (RLB) hybridization. In contrast, only 14.2% (23/162) and 1.2% (2/162) of clinical sputa were positive for E. dermatitidis and S. apiospermum / S. boydii species complex when tested by culture, respectively. Molecular detection methods, such as loop-mediated isothermal amplification (LAMP) or reverse line blot (RLB) hybridization, have the potential to become powerful alternatives to selective culture, providing a more realistic understanding on the prevalence of E. dermatitidis and S. apiospermum / S. boydii species complex in the respiratory tract of CF patients.

Multicenter Study of Method-Dependent Epidemiological Cutoff Values for Detection of Resistance in Candida spp. and Aspergillus spp. to Amphotericin B and Echinocandins for the Etest Agar Diffusion Method.

Method-dependent Etest epidemiological cutoff values (ECVs) are not available for susceptibility testing of either Candida or Aspergillus species with amphotericin B or echinocandins. In addition, reference caspofungin MICs for Candida spp. are unreliable. Candida and Aspergillus species wild-type (WT) Etest MIC distributions (microorganisms in a species-drug combination with no detectable phenotypic resistance) were established for 4,341 Candida albicans, 113 C. dubliniensis, 1,683 C. glabrata species complex (SC), 709 C. krusei, 767 C. parapsilosis SC, 796 C. tropicalis, 1,637 Aspergillus fumigatus SC, 238 A. flavus SC, 321 A. niger SC, and 247 A. terreus SC isolates. Etest MICs from 15 laboratories (in Argentina, Europe, Mexico, South Africa, and the United States) were pooled to establish Etest ECVs. Anidulafungin, caspofungin, micafungin, and amphotericin B ECVs (in micrograms per milliliter) encompassing ≥97.5% of the statistically modeled population were 0.016, 0.5, 0.03, and 1 for C. albicans; 0.03, 1, 0.03, and 2 for C. glabrata SC; 0.06, 1, 0.25, and 4 for C. krusei; 8, 4, 2, and 2 for C. parapsilosis SC; and 0.03, 1, 0.12, and 2 for C. tropicalis The amphotericin B ECV was 0.25 µg/ml for C. dubliniensis and 2, 8, 2, and 16 µg/ml for the complexes of A. fumigatus, A. flavus, A. niger, and A. terreus, respectively. While anidulafungin Etest ECVs classified 92% of the Candida fks mutants evaluated as non-WT, the performance was lower for caspofungin (75%) and micafungin (84%) cutoffs. Finally, although anidulafungin (as an echinocandin surrogate susceptibility marker) and amphotericin B ECVs should identify Candida and Aspergillus isolates with reduced susceptibility to these agents using the Etest, these ECVs will not categorize a fungal isolate as susceptible or resistant, as breakpoints do.

Somatic mutation, copy number and transcriptomic profiles of primary and matched metastatic estrogen receptor-positive breast cancers.

BACKGROUND: Estrogen receptor-positive (ER+) breast cancers (BCs) constitute the most frequent BC subtype. The molecular landscape of ER+ relapsed disease is not well characterized. In this study, we aimed to describe the genomic evolution between primary (P) and matched metastatic (M) ER+ BCs after failure of adjuvant therapy. MATERIALS AND METHODS: A total of 182 ER+ metastatic BC patients with long-term follow-up were identified from a single institution. P tumor tissue was available for all patients, with 88 having matched M material. According to the availability of tumor material, samples were characterized using a 120 mutational hotspot qPCR, a 29 gene copy number aberrations (CNA) and a 400 gene expression panels. ESR1 mutations were assayed by droplet digital PCR. Molecular alterations were correlated with overall survival (OS) using the Cox proportional hazards regression models. RESULTS: The median follow-up was 6.4 years (range 0.5-26.6 years). Genomic analysis of P tumors revealed somatic mutations in PIK3CA, KRAS, AKT1, FGFR3, HRAS and BRAF at frequencies of 41%, 6%, 5%, 2%, 1% and 2%, respectively, and CN amplification of CCND1, ZNF703, FGFR1, RSF1 and PAK1 at 23%, 19%, 17%, 12% and 11%, respectively. Mutations and CN amplifications were largely concordant between P and matched M (>84%). ESR1 mutations were found in 10.8% of the M but none of the P. Thirteen genes, among which ESR1, FOXA1, and HIF1A, showed significant differential expression between P and M. In P, the differential expression of 18 genes, among which IDO1, was significantly associated with OS (FDR < 0.1). CONCLUSIONS: Despite the large concordance between P and matched M for the evaluated molecular alterations, potential actionable targets such as ESR1 mutations were found only in M. This supports the importance of characterizing the M disease. Other targets we identified, such as HIF1A and IDO1, warrant further investigation in this patient population.

Phase I study of safety and pharmacokinetics of the anti-MUC16 antibody-drug conjugate DMUC5754A in patients with platinum-resistant ovarian cancer or unresectable pancreatic cancer.

BACKGROUND: MUC16 is a tumor-specific antigen overexpressed in ovarian (OC) and pancreatic (PC) cancers. The antibody-drug conjugate (ADC), DMUC5754A, contains the humanized anti-MUC16 monoclonal antibody conjugated to the microtubule-disrupting agent, monomethyl auristatin E (MMAE). PATIENTS AND METHODS: This phase I study evaluated safety, pharmacokinetics (PK), and pharmacodynamics of DMUC5754A given every 3 weeks (Q3W, 0.3-3.2 mg/kg) or weekly (Q1W, 0.8-1.6 mg/kg) to patients with advanced recurrent platinum-resistant OC or unresectable PC. Biomarker studies were also undertaken. RESULTS: Patients (66 OC, 11 PC) were treated with DMUC5754A (54 Q3W, 23 Q1W). Common related adverse events (AEs) in >20% of patients (all grades) over all dose levels were fatigue, peripheral neuropathy, nausea, decreased appetite, vomiting, diarrhea, alopecia, and pyrexia in Q3W patents, and nausea, vomiting, anemia, fatigue, neutropenia, alopecia, decreased appetite, diarrhea, and hypomagnesemia in Q1W patients. Grade ≥3-related AE in ≥5% of patients included neutropenia (9%) and fatigue (7%) in Q3W patients, and neutropenia (17%), diarrhea (9%), and hyponatremia (9%) in Q1W patients. Plasma antibody-conjugated MMAE (acMMAE) and serum total antibody exhibited non-linear PK across tested doses. Minimal accumulation of acMMAE, total antibody, or unconjugated MMAE was observed. Confirmed responses (1 CR, 6 PRs) occurred in OC patients whose tumors were MUC16-positive by IHC (2+ or 3+). Two OC patients had unconfirmed PRs; six OC patients had stable disease lasting >6 months. For CA125, a cut-off of ≥70% reduction was more suitable for monitoring treatment response due to the binding and clearance of serum CA125 by MUC16 ADC. We identified circulating HE4 as a potential novel surrogate biomarker for monitoring treatment response of MUC16 ADC and other anti-MUC16 therapies in OC. CONCLUSIONS: DMUC5754A has an acceptable safety profile and evidence of anti-tumor activity in patients with MUC16-expressing tumors. Objective responses were only observed in MUC16-high patients, although prospective validation is required. CLINICAL TRIAL NUMBER: NCT01335958.

Polymorphisms in Host Immunity-Modulating Genes and Risk of Invasive Aspergillosis: Results from the AspBIOmics Consortium.

Recent studies suggest that immune-modulating single-nucleotide polymorphisms (SNPs) influence the risk of developing cancer-related infections. Here, we evaluated whether 36 SNPs within 14 immune-related genes are associated with the risk of invasive aspergillosis (IA) and whether genotyping of these variants might improve disease risk prediction. We conducted a case-control association study of 781 immunocompromised patients, 149 of whom were diagnosed with IA. Association analysis showed that the IL4Rrs2107356 and IL8rs2227307 SNPs (using dbSNP numbering) were associated with an increased risk of IA (IL4Rrs2107356 odds ratio [OR], 1.92; 95% confidence interval [CI], 1.20 to 3.09; IL8rs2227307 OR, 1.73; 95% CI, 1.06 to 2.81), whereas the IL12Brs3212227 and IFNγrs2069705 variants were significantly associated with a decreased risk of developing the infection (IL12Brs3212227 OR, 0.60; 95% CI, 0.38 to 0.96; IFNγrs2069705 OR, 0.63; 95% CI, 0.41 to 0.97). An allogeneic hematopoietic stem cell transplantation (allo-HSCT)-stratified analysis revealed that the effect observed for the IL4Rrs2107356 and IFNγrs2069705 SNPs was stronger in allo-HSCT (IL4Rrs2107356 OR, 5.63; 95% CI, 1.20 to 3.09; IFNγrs2069705 OR, 0.24; 95% CI, 0.10 to 0.59) than in non-HSCT patients, suggesting that the presence of these SNPs renders patients more vulnerable to infection, especially under severe and prolonged immunosuppressive conditions. Importantly, in vitro studies revealed that carriers of the IFNγrs2069705C allele showed a significantly increased macrophage-mediated neutralization of fungal conidia (P = 0.0003) and, under stimulation conditions, produced higher levels of gamma interferon (IFNγ) mRNA (P = 0.049) and IFNγ and tumor necrosis factor alpha (TNF-α) cytokines (P value for 96 h of treatment with lipopolysaccharide [PLPS-96 h], 0.057; P value for 96 h of treatment with phytohemagglutinin [PPHA-96 h], 0.036; PLPS+PHA-96 h = 0.030; PPHA-72 h = 0.045; PLPS+PHA-72 h = 0.018; PLPS-96 h = 0.058; PLPS+PHA-96 h = 0.0058). Finally, we also observed that the addition of SNPs significantly associated with IA to a model including clinical variables led to a substantial improvement in the discriminatory ability to predict disease (area under the concentration-time curve [AUC] of 0.659 versus AUC of 0.564; P-2 log likehood ratio test = 5.2 · 10(-4) and P50.000 permutation test = 9.34 · 10(-5)). These findings suggest that the IFNγrs2069705 SNP influences the risk of IA and that predictive models built with IFNγ, IL8, IL12p70, and VEGFA variants can used to predict disease risk and to implement risk-adapted prophylaxis or diagnostic strategies.

Identification of Endoglin as an epigenetically regulated tumour-suppressor gene in lung cancer.

BACKGROUND: The transforming growth factor-beta (TGF- ß) pathway has been implicated in proliferation, migration and invasion of various cancers. Endoglin is a TGF-ß accessory receptor that modulates signalling. We identified Endoglin as an epigenetically silenced tumour-suppressor gene in lung cancer by means of a genome-wide screening approach, then sought to characterise its effect on lung cancer progression. METHODS: Methylation microarray and RNA sequencing were carried out on lung cancer cell lines. Epigenetic silencing of Endoglin was confirmed by methylation and expression analyses. An expression vector and a 20-gene expression panel were used to evaluate Endoglin function. Pyrosequencing was carried out on two independent cohorts comprising 112 and 202 NSCLC cases, respectively, and the impact of Endoglin methylation on overall survival (OS) was evaluated. RESULTS: Methylation in the promoter region resulted in silencing of Endoglin, which could be reactivated by demethylation. Increased invasion coupled with altered EMT marker expression was observed in cell lines with an epithelial-like, but not those with a mesenchymal-like, profile when Endoglin was absent. Methylation was associated with decreased OS in stage I but not in stages II-III disease. CONCLUSIONS: We show that Endoglin is a common target of epigenetic silencing in lung cancer. We reveal a link between Endoglin silencing and EMT progression that might be associated with decreased survival in stage I disease.

Evaluation of a modified EUCAST fragmented-mycelium inoculum method for in vitro susceptibility testing of dermatophytes and the activity of novel antifungal agents.

For antifungal susceptibility testing of nonsporulating or poorly sporulating dermatophytes, a fragmented-mycelium inoculum preparation method was established and compared to broth microdilution testing according to CLSI and EUCAST guidelines. Moreover, the in vitro activity of new antifungal agents against dermatophytes was evaluated. Agreement between the mycelial inoculum method and the CLSI broth microdilution method was high (93% to 100%). Echinocandins (minimal effective concentration [MEC], ≤0.5 mg/liter) and posaconazole (MIC, ≤3.00 mg/liter) showed good activity against all tested dermatophytes.

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.

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 ß-(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.

Susceptibility and diversity in the therapy-refractory genus scedosporium.

Scedosporium species show decreased susceptibility to the majority of systemic antifungal drugs. Acquired resistance is likely to disseminate differentially with the mode of exchange of genetic material between lineages. Inter- and intraspecific diversities of Scedosporium species were analyzed for three partitions (rDNA internal transcribed spacer gene [ITS], partial ß-tubulin gene, and amplified fragment length polymorphism profiles), with the aim to establish distribution of resistance between species, populations, and strains. Heterogeneity of and recombination between lineages were determined, and distances between clusters were calculated using a centroid approach. Clinical, geographic, and antifungal data were plotted on diversity networks. Scedosporium minutisporum, Scedosporium desertorum, and Scedosporium aurantiacum were distinguished unambiguously in all partitions and had differential antifungal susceptibility profiles (ASP). Pseudallescheria fusoidea and Pseudallescheria ellipsoidea were indistinguishable from Scedosporium boydii. Pseudallescheria angusta took an intermediate position between Scedosporium apiospermum and S. boydii. Scedosporium boydii and S. apiospermum had identical ASP. Differences in (multi)resistance were linked to individual strains. S. apiospermum and S. boydii showed limited interbreeding and were recognized as valid, sympatric species. The S. apiospermum/S. boydii group, comprising the main clinically relevant Scedosporium species, consists of separate lineages and is interpreted as a complex undergoing sympatric evolution with incomplete lineage sorting. In routine diagnostics, the lineages in S. apiospermum/S. boydii are indicated with the umbrella descriptor "S. apiospermum complex"; individual species can be identified with rDNA ITS with 96.3% confidence. Voriconazole is recommended as the first-line treatment; resistance against this compound is rare.

Changes in PIK3CA mutation status are not associated with recurrence, metastatic disease or progression in endocrine-treated breast cancer.

The phosphatidylinositol-3-kinase pathway plays an important role in proliferation, migration and survival in breast cancer and may play a role in resistance to endocrine therapy. Pathway activation occurs as a result of mutations in PIK3CA or loss of functional PTEN. Matched primary and recurrent samples from 120 breast cancer patients treated with endocrine therapy were profiled with a qPCR-based mutation assay covering eight mutational hotspots in PIK3CA. PTEN was assayed by immunohistochemistry. Samples were well characterized with respect to anatomic location of recurrence (metastatic nodal or local recurrence as opposed to contralateral or ipsilateral new primary cancers). In total, 43 % of patients had at least one PIK3CA mutation at diagnosis, and 41 % had a mutation at the time of recurrence. Only 8 % of patients with local recurrence, metastatic disease or progression on primary endocrine treatment changed their PIK3CA mutation status (four gains, two losses, total 76). The most common changes in PIK3CA mutation status were seen in patients who developed a new cancer either in the treated or contralateral breast (64 %, three gains, four losses, total 11). PIK3CA mutation status does not change in the majority of patients with breast cancer and the acquisition of mutations in PIK3CA is not responsible for the development of endocrine resistance. PTEN loss at diagnosis is associated with a significantly shorter time to progression compared with tumours in which PTEN was retained. These are the most comprehensive data currently available correlating PIK3CA status, site of recurrence and endocrine resistance.

In vitro activity of colistin as single agent and in combination with antifungals against filamentous fungi occurring in patients with cystic fibrosis.

Because published reports indicate that the antibiotic colistin (COL) has antifungal properties, this study investigated the antifungal in vitro activity of COL as single agent and in combination with the antifungal compounds voriconazole (VRC), caspofungin (CAS) and amphotericin B (AMB) against Scedosporium/Pseudallescheria spp., Exophiala dermatitidis and Geosmithia argillacea. In total, susceptibility was determined for 77 Scedosporium/Pseudallescheria spp., 82 E. dermatitidis and 17 G. argillacea isolates. The minimal inhibitory concentrations (MICs) of COL and the antifungals as single compound and in combination were determined with MIC test strips. Drug interactions were detected by crossing the MIC test strips at a 90º angle. The fractional inhibitory concentration index was used to categorise the drugs' interaction. The MIC50 value of COL was 12 µg ml(-1) for S. prolificans, 16 µg ml(-1) for P. apiosperma, 16 µg ml(-1) for P. boydii, 12 µg ml(-1) for E. dermatiditis and 6 µg ml(-1) for G. argillacea. VRC was the most active drug in combination without any antagonism with the exception of few P. boydii isolates. COL as single agent and in most combinations with antifungals exhibits in vitro antifungal activity against filamentous ascomycetes occurring in cystic fibrosis patients and may offer a novel therapeutic option, especially for multidrug-resistant S. prolificans.

Towards an automated data cleaning with deep learning in CRESST.

The CRESST experiment employs cryogenic calorimeters for the sensitive measurement of nuclear recoils induced by dark matter particles. The recorded signals need to undergo a careful cleaning process to avoid wrongly reconstructed recoil energies caused by pile-up and read-out artefacts. We frame this process as a time series classification task and propose to automate it with neural networks. With a data set of over one million labeled records from 68 detectors, recorded between 2013 and 2019 by CRESST, we test the capability of four commonly used neural network architectures to learn the data cleaning task. Our best performing model achieves a balanced accuracy of 0.932 on our test set. We show on an exemplary detector that about half of the wrongly predicted events are in fact wrongly labeled events, and a large share of the remaining ones have a context-dependent ground truth. We furthermore evaluate the recall and selectivity of our classifiers with simulated data. The results confirm that the trained classifiers are well suited for the data cleaning task.

Molecular identification tools for sibling species of Scedosporium and Pseudallescheria.

The aim of this study was to develop molecular identification tools for currently recognized species of Pseudallescheria and Scedosporium through the use of species-specific primers and RFLP, so as to enhance rapid differentiation of clinically relevant species. The variability of species was established in a set of 681 Internal Transcribed Spacer (ITS) and 349 ß-tubulin (BT2) sequences. Amplified Fragment Length Polymorphism profile clustering matched with BT2 results, whereas ITS grouping was less detailed. ITS was sufficient for the differentiation of most haplotypes of clinically relevant species (P. apiosperma, P. boydii, S. aurantiacum, S. dehoogii, and S. prolificans) and of environmental species (P. minutispora and Lophotrichus fimeti) when Restriction Fragment Length Polymorphism (RFLP) were applied. For the identification of P. apiosperma and P. boydii species-specific BT2 primers were needed. Pseudallescheria fusoidea, P. ellipsoidea and P. angusta remained difficult to distinguish from P. boydii.