Dea Garcia-Hermoso

A Global Analysis of Mucormycosis in France: The RetroZygo Study (2005–2007)

BACKGROUND Mucormycosis is a deadly invasive fungal infection whose characteristics are only partially understood. METHODS Data on mucormycosis obtained in France between 2005 and 2007 from 2 notification systems were merged. The 2008 European Organisation for Research and Treatment of Cancer/Mycoses Study Group definition criteria were applied and risk factors for death were analyzed by hazard ratios (HRs) calculated from the Cox proportional hazards regression model. RESULTS A total of 101 cases (60 proven, 41 probable), mostly in men (58%) >50 years (mean age, 50.7 ± 19.9) were recorded. Hematological malignancies represented 50% (median time for occurrence, 8.8 months after disease onset), diabetes 23%, and trauma 18% of cases. Sites of infection were lungs (28%; 79% in hematology patients), rhinocerebral (25%; 64% in diabetic patients), skin (20%), and disseminated (18%). Median time between first symptoms and diagnosis was 2 weeks. The main fungal species were Rhizopus oryzae (32%) and Lichtheimia species (29%). In cases where the causative species was identified, R. oryzae was present in 85% of rhinocerebral forms compared with only 17% of nonrhinocerebral forms (P < .001). Treatment consisted of surgery in 59% and antifungals in 87% of cases (liposomal amphotericin B in 61%). Ninety-day survival was 56%; it was reduced in cases of dissemination compared with rhinocerebral (HR, 5.38 [2.0-14.1]; P < .001), pulmonary (HR, 2.2 [1.0-4.7]; P = .04), or skin localization (HR, 5.73 [1.9-17.5]; P = .002); survival was reduced in cases of hematological malignancies compared with diabetes mellitus (HR, 2.3 [1.0-5.2]; P < .05) or trauma (HR, 6.9 [1.6-28.6], P = .008) and if ≥2 underlying conditions (HR, 5.9 [1.8-19.0]; P = .004). Mucormycosis localization remained the only independent factor associated with survival. CONCLUSIONS This 3-year study performed in one country shows the diverse clinical presentation of mucormycosis with a high prevalence of primary skin infection following trauma and a prognosis significantly influenced by localization.

Molecular Identification of Zygomycetes from Culture and Experimentally Infected Tissues

ABSTRACT Mucormycosis is an emerging infection associated with a high mortality rate. Identification of the causative agents remains difficult and time-consuming by standard mycological procedures. In this study, internal transcribed spacer (ITS) sequencing was validated as a reliable technique for identification of Zygomycetes to the species level. Furthermore, species identification directly from infected tissues was evaluated in experimentally infected mice. Fifty-four Zygomycetes strains belonging to 16 species, including the most common pathogenic species of Rhizopus spp., Absidia spp., Mucor spp., and Rhizomucor spp., were used to assess intra- and interspecies variability. Ribosomal DNA including the complete ITS1-5.8S-ITS2 region was amplified with fungal universal primers, sequenced, and compared. Overall, for a given species, sequence similarities between isolates were >98%. In contrast, ITS sequences were very different between species, allowing an accurate identification of Zygomycetes to the species level in most cases. Six species (Rhizopus oryzae, Rhizopus microsporus, Rhizomucor pusillus, Mucor circinelloides, and Mucor indicus) were also used to induce disseminated mucormycosis in mice and to demonstrate that DNA extraction, amplification of fungal DNA, sequencing, and molecular identification were possible directly from frozen tissues.

One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes

The aim of this study was to assess potential candidate gene regions and corresponding universal primer pairs as secondary DNA barcodes for the fungal kingdom, additional to ITS rDNA as primary barcode. Amplification efficiencies of 14 (partially) universal primer pairs targeting eight genetic markers were tested across > 1 500 species (1 931 strains or specimens) and the outcomes of almost twenty thousand (19 577) polymerase chain reactions were evaluated. We tested several well-known primer pairs that amplify: i) sections of the nuclear ribosomal RNA gene large subunit (D1–D2 domains of 26/28S); ii) the complete internal transcribed spacer region (ITS1/2); iii) partial β -tubulin II (TUB2); iv) γ-actin (ACT); v) translation elongation factor 1-α (TEF1α); and vi) the second largest subunit of RNA-polymerase II (partial RPB2, section 5–6). Their PCR efficiencies were compared with novel candidate primers corresponding to: i) the fungal-specific translation elongation factor 3 (TEF3); ii) a small ribosomal protein necessary for t-RNA docking; iii) the 60S L10 (L1) RP; iv) DNA topoisomerase I (TOPI); v) phosphoglycerate kinase (PGK); vi) hypothetical protein LNS2; and vii) alternative sections of TEF1α. Results showed that several gene sections are accessible to universal primers (or primers universal for phyla) yielding a single PCR-product. Barcode gap and multi-dimensional scaling analyses revealed that some of the tested candidate markers have universal properties providing adequate infra- and inter-specific variation that make them attractive barcodes for species identification. Among these gene sections, a novel high fidelity primer pair for TEF1α, already widely used as a phylogenetic marker in mycology, has potential as a supplementary DNA barcode with superior resolution to ITS. Both TOPI and PGK show promise for the Ascomycota, while TOPI and LNS2 are attractive for the Pucciniomycotina, for which universal primers for ribosomal subunits often fail.

Candida spp. with Acquired Echinocandin Resistance, France, 2004–2010

We report 20 episodes of infection caused by acquired echinocandin-resistant Candida spp. harboring diverse and new Fksp mutations. For 12 patients, initial isolates (low MIC, wild-type Fksp sequence) and subsequent isolates (after caspofungin treatment, high MIC, mutated Fksp) were genetically related.

High prevalence of triazole resistance in Aspergillus fumigatus, especially mediated by TR/L98H, in a French cohort of patients with cystic fibrosis

OBJECTIVES Triazole resistance in Aspergillus fumigatus due to a single azole resistance mechanism (TR/L98H) is increasingly reported in European countries. Data from patients with cystic fibrosis (CF) are limited. Our study aimed to investigate the prevalence and molecular mechanisms of azole resistance in A. fumigatus in a cohort of patients with CF. METHODS Eighty-five A. fumigatus isolates from 50 CF patients, collected between January 2010 and April 2011, were retrospectively analysed for azole resistance using agar plates containing 4 mg/L itraconazole. MICs of itraconazole, voriconazole and posaconazole were determined according to EUCAST methodology for each isolate able to grow on this medium. Species identification was performed by sequencing of the β-tubulin gene. Sequencing analysis of the cyp51A gene and its promoter region was conducted. RESULTS Nine isolates (four patients, 8% prevalence) were able to grow on itraconazole-containing agar plates. Itraconazole resistance was confirmed by EUCAST methodology (MICs >2 mg/L). All isolates had mutations in the cyp51A gene at residues previously involved in azole resistance: L98H (n = 5), M220T (n = 4) and G54R (n = 1). One patient had three genetically distinct azole-resistant isolates identified during the study. The isolates with L98H that were recovered from three patients (6% prevalence) also had the 34 bp tandem repeat in the promoter region of cyp51A (TR/L98H) and displayed multiazole resistance. CONCLUSIONS We report an 8% prevalence of itraconazole resistance in CF patients in our centre, mostly driven by TR/L98H (6%). Our data confirm that TR/L98H occurs in France and can be highly prevalent in CF patients.

Proposed nomenclature for Pseudallescheria, Scedosporium and related genera

As a result of fundamental changes in the International Code of Nomenclature on the use of separate names for sexual and asexual stages of fungi, generic names of many groups should be reconsidered. Members of the ECMM/ISHAM working group on Pseudallescheria/Scedosporium infections herein advocate a novel nomenclature for genera and species in Pseudallescheria, Scedosporium and allied taxa. The generic names Parascedosporium, Lomentospora, Petriella, Petriellopsis, and Scedosporium are proposed for a lineage within Microascaceae with mostly Scedosporium anamorphs producing slimy, annellidic conidia. Considering that Scedosporium has priority over Pseudallescheria and that Scedosporium prolificans is phylogenetically distinct from the other Scedosporium species, some name changes are proposed. Pseudallescheria minutispora and Petriellidium desertorum are renamed as Scedosporium minutisporum and S. desertorum, respectively. Scedosporium prolificans is renamed as Lomentospora prolificans.

Mixed Infections and In Vivo Evolution in the Human Fungal Pathogen Cryptococcus neoformans

ABSTRACT Koch’s postulates are criteria establishing a causal relationship between a microbe and a disease that lead to the assumption that diseases are caused by a single strain or its evolved forms. Cryptococcus neoformans is a life-threatening human fungal pathogen responsible for an estimated 1 million cases of cryptococcosis/year, predominantly meningoencephalitis. To assess the molecular diversity of clinical isolates and gain knowledge of C. neoformans biology in the host, we analyzed clinical cultures collected during the prospective CryptoA/D study. Using molecular analysis of unpurified isolates, we demonstrated that mixed infections in humans are more common than previously thought, occurring in almost 20% of patients diagnosed with cryptococcosis. These mixed infections are composed of different mating types, serotypes, and/or genotypes. We also identified genetically related haploid and diploid strains in the same patients. Experimental infections and quantitative PCR show that these ploidy changes can result from endoreplication (duplication of DNA content) and that shuttling between haploid and diploid states can occur, suggesting in vivo evolution. Thus, the concept of one strain/one infection does not hold true for C. neoformans and may apply to other environmentally acquired fungal pathogens. Furthermore, the possibility of mixed and/or evolving infections should be taken into account when developing therapeutic strategies against these pathogens. IMPORTANCECryptococcus  neoformans is a life-threatening human fungal pathogen that is present in the environment and is responsible for an estimated 1 million cases of cryptococcosis/year, predominantly meningoencephalitis in HIV-infected patients. To assess the molecular diversity of clinical isolates and gain knowledge of C. neoformans biology in the host, we analyzed clinical cultures collected during a prospective study on cryptococcosis. Using molecular analysis of unpurified isolates, we uncovered an unexpectedly high frequency (almost 20%) of mixed infections. We further demonstrated that these mixed infections could result from infestation by multiple strains acquired from the environment. We also made the serendipitous discovery of in vivo evolution leading to endoreplication of the yeasts within the host. Thus, the concept of one strain causing one infection does not hold true for C. neoformans and potentially for other environmentally acquired fungal pathogens. The possibility of mixed and/or evolving infections should be taken into account when developing therapeutic strategies against these pathogens. Cryptococcus  neoformans is a life-threatening human fungal pathogen that is present in the environment and is responsible for an estimated 1 million cases of cryptococcosis/year, predominantly meningoencephalitis in HIV-infected patients. To assess the molecular diversity of clinical isolates and gain knowledge of C. neoformans biology in the host, we analyzed clinical cultures collected during a prospective study on cryptococcosis. Using molecular analysis of unpurified isolates, we uncovered an unexpectedly high frequency (almost 20%) of mixed infections. We further demonstrated that these mixed infections could result from infestation by multiple strains acquired from the environment. We also made the serendipitous discovery of in vivo evolution leading to endoreplication of the yeasts within the host. Thus, the concept of one strain causing one infection does not hold true for C. neoformans and potentially for other environmentally acquired fungal pathogens. The possibility of mixed and/or evolving infections should be taken into account when developing therapeutic strategies against these pathogens.

Cryptococcus neoformans capsule structure evolution in vitro and during murine infection.

ABSTRACT Cryptococcus neoformans capsule structure modifications after prolonged in vitro growth or in vivo passaging have been reported previously. However, nothing is known about the dynamics of these modifications or about their environmental specificities. In this study, capsule structure modifications after mouse passaging and prolonged in vitro culturing were analyzed by flow cytometry using the glucuronoxylomannan-specific monoclonal antibody E1. The capsule structures of strains recovered after 0, 1, 8, and 35 days were compared by using the level of E1-specific epitope expression and its cell-to-cell heterogeneity within a given cell population. In vitro, according to these parameters, the diversity of the strains was higher on day 35 than it was initially, suggesting the absence of selection during in vitro culturing. In contrast, the diversity of the strains recovered from the brain tended to decrease over time, suggesting that selection of more adapted strains had occurred. The strains recovered on day 35 from the spleen and the lungs had different phenotypes than the strains isolated from the brain of the same mouse on the same day, thus strongly suggesting that there is organ specificity for C. neoformans strain selection. Fingerprinting of the strains recovered in vitro and in vivo over time confirmed that genotypes evolved very differently in vitro and in vivo, depending on the environment. Overall, our results suggest that organ-specific selection can occur during cryptococcosis.

Molecular Phylogeny and Proposal of Two New Species of the Emerging Pathogenic Fungus Saksenaea

ABSTRACT Saksenaea is a monotypic genus belonging to the order Mucorales and capable of producing severe human infections. Through a polyphasic study based on analysis of the sequences of the internal transcribed spacer (ITS) region, domains D1 and D2 of the 28S rRNA gene, and the elongation factor 1α (EF-1α) gene, as well as by evaluation of relevant morphological and physiological characteristics of a set of clinical and environmental strains, we have demonstrated that Saksenaea vasiformis is a complex of species. We propose as new species Saksenaea oblongispora, characterized by oblong sporangiospores and unable to grow at 42°C, and Saksenaea erythrospora, characterized by large sporangiophores and sporangia and by ellipsoid sporangiospores, biconcave in the lateral view. Itraconazole, posaconazole, and terbinafine were active against all isolates included in the study, while amphotericin B, voriconazole, and the echinocandins showed low activity.

Molecular and Phenotypic Evaluation of Lichtheimia corymbifera (Formerly Absidia corymbifera) Complex Isolates Associated with Human Mucormycosis: Rehabilitation of L. ramosa

ABSTRACT Thirty-eight isolates (including 28 isolates from patients) morphologically identified as Lichtheimia corymbifera (formerly Absidia corymbifera) were studied by sequence analysis (analysis of the internal transcribed spacer [ITS] region of the ribosomal DNA, the D1-D2 region of 28S, and a portion of the elongation factor 1α [EF-1α] gene). Phenotypic characteristics, including morphology, antifungal susceptibility, and carbohydrate assimilation, were also determined. Analysis of the three loci uncovered two well-delimited clades. The maximum sequence similarity values between isolates from both clades were 66, 95, and 93% for the ITS, 28S, and EF-1α loci, respectively, with differences in the lengths of the ITS sequences being detected (763 to 770 bp for isolates of clade 1 versus 841 to 865 bp for isolates of clade 2). Morphologically, the shapes and the sizes of the sporangiospores were significantly different among the isolates from both clades. On the basis of the molecular and morphological data, we considered isolates of clade 2 to belong to a different species named Lichtheimia ramosa because reference strains CBS 269.65 and CBS 270.65 (which initially belonged to Absidia ramosa) clustered within this clade. As neotype A. corymbifera strain CBS 429.75 belongs to clade 1, the name L. corymbifera was conserved for clade 1 isolates. Of note, the amphotericin B MICs were significantly lower for L. ramosa than for L. corymbifera (P < 0.005) but were always ≤0.5 μg/ml for both species. Among the isolates tested, the assimilation of melezitose was positive for 67% of the L. ramosa isolates and negative for all L. corymbifera isolates. In conclusion, this study reveals that two Lichtheimia species are commonly associated with mucormycosis in humans.