Identification and Characterization of Mediators of Fluconazole Tolerance in Candida albicans.

Candida albicans is an important human pathogen and a major concern in intensive care units around the world. C. albicans infections are associated with a high mortality despite the use of antifungal treatments. One of the causes of therapeutic failures is the acquisition of antifungal resistance by mutations in the C. albicans genome. Fluconazole (FLC) is one of the most widely used antifungal and mechanisms of FLC resistance occurring by mutations have been extensively investigated. However, some clinical isolates are known to be able to survive at high FLC concentrations without acquiring resistance mutations, a phenotype known as tolerance. Mechanisms behind FLC tolerance are not well studied, mainly due to the lack of a proper way to identify and quantify tolerance in clinical isolates. We proposed here culture conditions to investigate FLC tolerance as well as an easy and efficient method to identity and quantify tolerance to FLC. The screening of C. albicans strain collections revealed that FLC tolerance is pH- and strain-dependent, suggesting the involvement of multiple mechanisms. Here, we addressed the identification of FLC tolerance mediators in C. albicans by an overexpression strategy focusing on 572 C. albicans genes. This strategy led to the identification of two transcription factors, CRZ1 and GZF3. CRZ1 is a C2H2-type transcription factor that is part of the calcineurin-dependent pathway in C. albicans, while GZF3 is a GATA-type transcription factor of unknown function in C. albicans. Overexpression of each gene resulted in an increase of FLC tolerance, however, only the deletion of CRZ1 in clinical FLC-tolerant strains consistently decreased their FLC tolerance. Transcription profiling of clinical isolates with variable levels of FLC tolerance confirmed a calcineurin-dependent signature in these isolates when exposed to FLC

Matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry for fast and accurate identification of clinically relevant Aspergillus species

AbstractNew Aspergillus species have recently been described with the use of multilocus sequencing in refractory cases of invasive aspergillosis. The classical phenotypic identification methods routinely used in clinical laboratories failed to identify them adequately. Some of these Aspergillus species have specific patterns of susceptibility to antifungal agents, and misidentification may lead to inappropriate therapy. We developed a matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) mass spectrometry (MS)‐based strategy to adequately identify Aspergillus species to the species level. A database including the reference spectra of 28 clinically relevant species from seven Aspergillus sections (five common and 23 unusual species) was engineered. The profiles of young and mature colonies were analysed for each reference strain, and species‐specific spectral fingerprints were identified. The performance of the database was then tested on 124 clinical and 16 environmental isolates previously characterized by partial sequencing of the β‐tubulin and calmodulin genes. One hundred and thirty‐eight isolates of 140 (98.6%) were correctly identified. Two atypical isolates could not be identified, but no isolate was misidentified (specificity: 100%). The database, including species‐specific spectral fingerprints of young and mature colonies of the reference strains, allowed identification regardless of the maturity of the clinical isolate. These results indicate that MALDI‐TOF MS is a powerful tool for rapid and accurate identification of both common and unusual species of Aspergillus. It can give better results than morphological identification in clinical laboratories

Epidemiology of invasive candidiasis in a surgical intensive care unit: an observational study

Background: Invasive candidiasis (IC) is a frequent and life-threatening infection in critically ill patients. The aim of this study was to evaluate the epidemiology of IC and the antifungal susceptibility of etiological agents in patients admitted to our surgical intensive care unit (SICU) in Spain. Methods: We designed a prospective, observational, single center, population-based study in a SICU. We included all consecutive adult patients (≥18 years old) who had documented IC, either on admission or during their stay, between January 2012 and December 2013. Results: There were a total of 22 episodes of IC in the 1149 patients admitted during the 24-month study. The overall IC incidence was 19.1 cases per 1000 admissions. Thirteen cases of IC (59.1 %) were intra-abdominal candidiasis (IAC) and 9 (40.9 %) were candidemias. All cases of IAC were patients with secondary peritonitis and severe sepsis or septic shock. The overall crude mortality rate was 13.6 %; while, it was 33 % in patients with candidemia. All patients with IAC survived, including one patient with concomitant candidemia. The most common species causing IC was Candida albicans (13; 59.1 %) followed by Candida parapsilosis (5; 22.7 %), and Candida glabrata (2; 9.1 %). There was also one case each (4.5 %) of Candida krusei and Candida tropicalis. Thus, the ratio of non-C. albicans (9) to C. albicans (13) was 1:1.4. There was resistance to fluconazole and itraconazole in 13.6 % of cases. Resistance to other antifungals was uncommon. Conclusions: Candida parapsilosis was the second most common species after C. albicans, indicating the high prevalence of non-C. albicans species in the SICU. Resistance to azoles, particularly fluconazole, should be considered when starting an empirical treatment. Although IAC is a very frequent form of IC in critically ill surgical patients, prompt antifungal therapy and adequate source control appears to lead to a good outcome. However, our results are closely related to our ICU and any generalization must be taken with caution. Therefore, further investigations are needed. Keywords: Intensive care unit, Invasive candidiasis, Candidemia, Antifungal susceptibilit

Comparing genomic variant identification protocols for Candida auris.

Genomic analyses are widely applied to epidemiological, population genetic and experimental studies of pathogenic fungi. A wide range of methods are employed to carry out these analyses, typically without including controls that gauge the accuracy of variant prediction. The importance of tracking outbreaks at a global scale has raised the urgency of establishing high-accuracy pipelines that generate consistent results between research groups. To evaluate currently employed methods for whole-genome variant detection and elaborate best practices for fungal pathogens, we compared how 14 independent variant calling pipelines performed across 35 Candida auris isolates from 4 distinct clades and evaluated the performance of variant calling, single-nucleotide polymorphism (SNP) counts and phylogenetic inference results. Although these pipelines used different variant callers and filtering criteria, we found high overall agreement of SNPs from each pipeline. This concordance correlated with site quality, as SNPs discovered by a few pipelines tended to show lower mapping quality scores and depth of coverage than those recovered by all pipelines. We observed that the major differences between pipelines were due to variation in read trimming strategies, SNP calling methods and parameters, and downstream filtration criteria. We calculated specificity and sensitivity for each pipeline by aligning three isolates with chromosomal level assemblies and found that the GATK-based pipelines were well balanced between these metrics. Selection of trimming methods had a greater impact on SAMtools-based pipelines than those using GATK. Phylogenetic trees inferred by each pipeline showed high consistency at the clade level, but there was more variability between isolates from a single outbreak, with pipelines that used more stringent cutoffs having lower resolution. This project generated two truth datasets useful for routine benchmarking of C. auris variant calling, a consensus VCF of genotypes discovered by 10 or more pipelines across these 35 diverse isolates and variants for 2 samples identified from whole-genome alignments. This study provides a foundation for evaluating SNP calling pipelines and developing best practices for future fungal genomic studies

Horizontal Transmission of Candida albicans and Evidence of a Vaccine Response in Mice Colonized with the Fungus

Disseminated candidiasis is the third leading nosocomial blood stream infection in the United States and is often fatal. We previously showed that disseminated candidiasis was preventable in normal mice by immunization with either a glycopeptide or a peptide synthetic vaccine, both of which were Candida albicans cell wall derived. A weakness of these studies is that, unlike humans, mice do not have a C. albicans GI flora and they lack Candida serum antibodies. We examined the influence of C. albicans GI tract colonization and serum antibodies on mouse vaccination responses to the peptide, Fba, derived from fructose bisphosphate aldolase which has cytosolic and cell wall distributions in the fungus. We evaluated the effect of live C. albicans in drinking water and antimicrobial agents on establishment of Candida colonization of the mouse GI tract. Body mass, C. albicans in feces, and fungal-specific serum antibodies were monitored longitudinally. Unexpectedly, C. albicans colonization occurred in mice that received only antibiotics in their drinking water, provided that the mice were housed in the same room as intentionally colonized mice. The fungal strain in unintentionally colonized mice appeared identical to the strain used for intentional GI-tract colonization. This is the first report of horizontal transmission and spontaneous C. albicans colonization in mice. Importantly, many Candida-colonized mice developed serum fungal-specific antibodies. Despite the GI-tract colonization and presence of serum antibodies, the animals made antibodies in response to the Fba immunogen. This mouse model has potential for elucidating C. albicans horizontal transmission and for exploring factors that induce host defense against disseminated candidiasis. Furthermore, a combined protracted GI-tract colonization with Candida and the possibility of serum antibody responses to the presence of the fungus makes this an attractive mouse model for testing the efficacy of vaccines designed to prevent human disseminated candidiasis

Sequential or alternating administration of docetaxel (Taxotere®) combined with FEC in metastatic breast cancer: a randomised phase II trial

The aim of this study, using a Fleming single-stage design, was to explore the efficacy and safety of Taxotere® 100 mg m−2 docetaxel and FEC 75 cyclophosphamide 500 mg m−2, fluorouracil 500 mg m−2 and epirubicin 75 mg m−2, in alternating and sequential schedules for the first-line treatment of metastatic breast cancer. One hundred and thirty-six women were randomly allocated, to one of three treatment regimens: DTX 100 plus FEC 75, alternated for eight courses (ALT); four courses of DTX 100 followed by four courses of FEC 75 (SEQ T); or four courses of FEC 75 followed by four courses of DTX 100 (SEQ F). One hundred and thirty-one women were evaluable for tumour response. Although the treatment outcome was equivalent in the two sequential arms and the alternating regimen (P=0.110, not significant), the response rate was less encouraging in the SEQ F arm (52.3%) than in the other two arms (71.1% for ALT and 70.5% for SEQ T), in which docetaxel was administered first. Time to progression was similar in the ALT, SEQ T and SEQ F arms (9.5, 9.3 and 10.4 months respectively). Grade 3–4 neutropenia was observed in nearly all patients; febrile neutropenia occurred in 9% (ALT), 16% (SEQ T) and 2% (SEQ F) of patients. Few patients (⩽9%) developed grade 3–4 non-haematological toxicities. Relative dose intensity was 97–99% for all regimens. All treatment regimens were active and well tolerated

A CO2 sensing module modulates β-1,3-glucan exposure in Candida albicans.

This work was funded by a program grant to A.J.P.B., N.A.R.G., L.P.E., and M.G.N. from the UK Medical Research Council [www.mrc.ac.uk: MR/M026663/1, MR/M026663/2]. The work was also supported by the Medical Research Council Centre for Medical Mycology [MR/N006364/1, MR/N006364/2], by a grant to C.d.E. from the European Commission [FunHoMic: H2020-MSCA-ITN-2018–812969], and by the Wellcome Trust via Investigator, Collaborative, Equipment, Strategic and Biomedical Resource awards [www.wellcome.ac.uk: 075470, 086827, 093378, 097377, 099197, 101873, 102705, 200208, 217163, 224323]. Work in the d’Enfert laboratory was supported by grants from the Agence Nationale de Recherche (ANR-10-LABX-62-IBEID) and the Swiss National Science Foundation (Sinergia CRSII5_173863/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.Peer reviewedPublisher PD

Travelers With Cutaneous Leishmaniasis Cured Without Systemic Therapy

Guidelines recommend wound care and/or local therapy as first-line treatment for cutaneous leishmaniasis. An analysis of a referral treatment program in 135 travelers showed that this approach was feasible in 62% of patients, with positive outcome in 83% of evaluable patient