Showing papers in "Medical Mycology in 2014"

Identification of filamentous fungi isolates by MALDI-TOF mass spectrometry: clinical evaluation of an extended reference spectra library.

Abstract: The identification of filamentous fungi by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) relies mainly on a robust and extensive database of reference spectra. To this end, a large in-house library containing 760 strains and representing 472 species was built and evaluated on 390 clinical isolates by comparing MALDI-TOF MS with the classical identification method based on morphological observations. The use of MALDI-TOF MS resulted in the correct identification of 95.4% of the isolates at species level, without considering LogScore values. Taking into account the Brukers' cutoff value for reliability (LogScore >1.70), 85.6% of the isolates were correctly identified. For a number of isolates, microscopic identification was limited to the genus, resulting in only 61.5% of the isolates correctly identified at species level while the correctness reached 94.6% at genus level. Using this extended in-house database, MALDI-TOF MS thus appears superior to morphology in order to obtain a robust and accurate identification of filamentous fungi. A continuous extension of the library is however necessary to further improve its reliability. Indeed, 15 isolates were still not represented while an additional three isolates were not recognized, probably because of a lack of intraspecific variability of the corresponding species in the database.

New insights into innate immune control of systemic candidiasis.

Abstract: Systemic infection caused by Candida species is the fourth leading cause of nosocomial bloodstream infection in modern hospitals and carries high morbidity and mortality despite antifungal therapy. A recent surge of immunological studies in the mouse models of systemic candidiasis and the parallel discovery and phenotypic characterization of inherited genetic disorders in antifungal immune factors that are associated with enhanced susceptibility or resistance to the infection have provided new insights into the cellular and molecular basis of protective innate immune responses against Candida. In this review, the new developments in our understanding of how the mammalian immune system responds to systemic Candida challenge are synthesized and important future research directions are highlighted.

Recognizing filamentous basidiomycetes as agents of human disease: A review

Abstract: Filamentous basidiomycetes (BM) are common environmental fungi that have recently emerged as important human pathogens, inciting a wide array of clinical manifestations that include allergic and invasive diseases. We reviewed 218 reported global cases of BM fungi. The most common etiologic agent was Schizophyllum commune in 52.3% (114/218) of the cases followed by Hormographiella aspergillata (n = 13; 5.9%), Ceriporia lacerata (n = 11; 5%), and, rarely, Volvariella volvacea, Inonotus tropicalis, Irpex lacteus, Phellinus undulates, Perenniporia species, Bjerkandera adusta, Sporotrichum pruinosum, Phanerochaete steroids, and Cyclomyces tabacinus. These fungi are present in the environment as gilled mushrooms, shelf fungi, and bracket fungi. However, in clinical settings, they usually present as nonsporulating white moulds that are difficult to identify. Moreover, the GenBank database of these fungi is limited. Regarding the country-wise distribution of cases, Japan topped the list with about 43% (n = 94) of globally reported cases, followed by India (57; 26%), the United States (4%), Austria (3.2%), Iran (3.2%), France (2.8%), and the remaining one-third from 16 other countries. The respiratory tract was the most commonly afflicted site (n = 71), with the majority of the cases (42; 59.1%) being allergic in etiology and comprising 34 cases of allergic bronchopulmonary mycosis. Also, B. adusta has been implicated in a recently described clinical entity, that is, fungus associated chronic cough, reported exclusively from Japan. BM fungi-incited diseases are currently underdiagnosed due to lack of awareness and expertise, warranting comprehensive epidemiological and susceptibility studies to determine their prevalence and to predict a more appropriate therapy.

Bloodstream infections by Malassezia and Candida species in critical care patients.

Abstract: Despite being considered an emerging yeast related to immunocompromised individuals, severe infections by Malassezia furfur have not been evaluated. During a one-year survey on yeasts fungemia, 290 neonatal and 17 pediatric patients with intravascular catheters, lipid parenteral nutrition, prolonged ward stay, and surgery were enrolled. In addition, the origin of the infection was investigated by swabbing hand skin of patients, parents, and healthcare workers and medical devices. All biological specimens and swabs were cultured on Sabouraud dextrose agar and Dixon agar. The yeasts identification was based on morphological and biochemical features and by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and confirmed by sequencing the internal transcribed spacer of nuclear ribosomal DNA. A higher prevalence of M. furfur (2.1%) over Candida spp. (1.4%) caused bloodstream infections (BSIs). Twelve fungemia episodes were recorded: 2 by M. furfur in a pediatric ward and 10 in a neonatal intensive care unit (6 caused by M. furfur and 4 by Candida spp.). M. furfur was also isolated from the skin of all patients with BSIs, from the hand skin of a parent, and from an incubator surface and sheet. Patients with Candida spp. and M. furfur BSIs were successfully treated with intravenous liposomal Amphotericin B. These findings highlight the need for a more accurate etiological diagnosis in high-risk patients by adding lipid-supplemented culture media for Malassezia in the current mycological

Characterization of Alternaria infectoria extracellular vesicles.

Abstract: Many fungi use membrane vesicles to transport complex molecules across their cell walls. Like mammalian exosomes, fungal vesicles contain lipids, proteins, and polysaccharides, many of which are associated with virulence. Here we identify and characterize extracellular vesicles (EVs) in Alternaria infectoria, a ubiquitous, environmental filamentous fungus that is also an opportunistic human pathogen. Examination of the A. infectoria EVs revealed a morphology similar to that of vesicles described in other fungal species. Of note, proteomic analysis detected a reduced number of vesicle-associated proteins. There were two prevalent categories among the 20 identified proteins, including the polysaccharide metabolism group, probably related to plant host invasion or biosynthesis/degradation of cell wall components, and the nuclear proteins, especially DNA repair enzymes. We also found enzymes related to pigment synthesis, adhesion to the host cell, and trafficking of vesicles/organelles/molecules. This is the first time EV secretions have been identified in a filamentous fungus. We believe that these vesicles might have a role in virulence.

Antifungal susceptibility of Malassezia furfur, Malassezia sympodialis, and Malassezia globosa to azole drugs and amphotericin B evaluated using a broth microdilution method

Abstract: We studied the in vitro activity of fluconazole (FCZ), ketoconazole (KTZ), miconazole (MCZ), voriconazole (VCZ), itraconazole (ITZ) and amphotericin B (AMB) against the three major pathogenicMalasseziaspecies,M. globosa,M. sympodialis,andM. furfur. Antifungal susceptibilities were determined using the broth microdilution method in accordance with Clinical and Laboratory Standards Institute reference document M27-A3. To support lipid-dependent yeast development, glucose, peptone, ox bile, malt extract, glycerol, and Tween supplements were added to Roswell Park Memorial Institute RPMI 1640 medium. The supplemented medium allowed good growth of all three species studied. The minimal inhibitory concentrations (MICs) were recorded after 72 h of incubation at 32 o C. The three species showed different susceptibility profiles for the drugs tested. Malassezia sympodialis was the most susceptible and M. furfur the least susceptible species. KTZ, ITZ, and VCZ were the most active drugs, showing low variability among isolates of the same species. FCZ, MCZ, and AMB showed high MICs and wide MIC ranges. Differences observed emphasize the need to accurately identify and evaluate antifungal susceptibility of Malassezia species. Further investigations and collaborative studies are essential for correlating in vitro results with clinical outcomes since the existing limited data do not allow definitive conclusions.

Interindividual variability and intraindividual stability of oral fungal microbiota over time

Abstract: Oral microbiota is one of the most complex and diverse microbial communities in the human body. In the present study, we aimed to characterize oral fungi biodiversity and stability over time in a group of healthy participants with good oral health. Oral health and oral fungal microbiota were evaluated in 40 healthy individuals. A follow-up of 10 participants was carried out 28 weeks and 30 weeks after the first sampling. Oral rinse was collected and incubated in a fungal selective medium at 25oC and 37oC for 7 days. Fungi were identified based on macro- and microscopic morphology. API/ID32C was used for yeast identification, and molecular techniques were used to identify the most prevalent nonidentified moulds, mainly by sequencing 18S and internally transcribed spacer regions. Moulds were recovered from all participants and yeast from 92.5%. The most frequently isolated fungi were Candida spp., Rhodotorula spp., Penicillium spp., Aspergillus spp., and Cladosporium spp. The oral fungal community presented a high interindividual variability, but the frequency and quantification of each fungal taxon was constant over the 30-week observation period, showing a consistent intraindividual stability over time. The intraindividual stability opposed to interindividual variability may suggest a common and a variable group of fungi in the oral cavity.

Molecular screening of 246 Portuguese Aspergillus isolates among different clinical and environmental sources

Abstract: Clinical and environmental samples from Portugal were screened for the presence of Aspergillus and the distributions of the species complexes were determined in order to understand how their distributions differ based on their source. Fifty-seven Aspergillus isolates from clinical samples were collected from 10 health institutions. Six species complexes were detected by internal transcribed spacer sequencing; Fumigati, Flavi ,a nd Nigri were found most frequently (50.9%, 21.0%, and 15.8%, respectively). β-tubulin and calmodulin sequencing resulted in seven cryptic species (A. awamorii, A. brasiliensis, A. fructus, A. lentulus, A. sydowii, A. tubingensis, Emericella echinulata) being identified among the 57 isolates. Thirty-nine isolates of Aspergillus were recovered from beach sand and poultry farms, 31 from swine farms, and 80 from hospital environments, for a total 189 isolates. Eleven species complexes were found in these 189 isolates, and those belonging to the Versicolores species complex were found most frequently (23.8%). There was a significant association between the different environmental sources and distribution of the species complexes; the hospital environment had greater variability of species complexes than other environmental locations. A high prevalence of cryptic species within the Circumdati complex was detected in several environments; from the isolates analyzed, at least four cryptic species were identified, most of them growing at 37 o C. Because Aspergillus species complexes have different susceptibilities to antifungals, knowing the species-complex epidemiology for each setting, as well as the identification of cryptic species among the collected clinical isolates, is important. This

Uncommon opportunistic yeast bloodstream infections from Qatar

Abstract: Eleven uncommon yeast species that are associated with high mortality rates irrespective of antifungal therapy were isolated from 17/187 (201 episodes) pediatric and elderly patients with fungemia from Qatar. The samples were taken over a 6-year period (January 2004-December 2010). Isolated species included Kluyveromyces marxianus, Lodderomyces elongisporus, Lindnera fabianii, Candida dubliniensis, Meyerozyma guilliermondii, Candida intermedia, Pichia kudriavzevii, Yarrowia lipolytica, Clavispora lusitaniae, Candida pararugosa, and Wickerhamomyces anomalus. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry provided correct identifications compared with molecular analysis testing of the same isolates. Low minimal inhibitory concentrations were found when isavuconazole and voriconazole were used for all uncommon yeast species evaluated in this study. Resistance to antifungal drugs was low and remained restricted to a few species.

Nucleotide sequence analysis of beta tubulin gene in a wide range of dermatophytes

Abstract: We investigated the resolving power of the beta tubulin protein-coding gene (BT2) for systematic study of dermatophyte fungi. Initially, 144 standard and clinical strains belonging to 26 species in the genera Trichophyton, Microsporum, and Epidermophyton were identified by internal transcribe spacer (ITS) sequencing. Subsequently, BT2 was partially amplified in all strains, and sequence analysis performed after construction of a BT2 database that showed length ranged from approximately 723 (T. ajelloi) to 808 nucleotides (M. persicolor) in different species. Intraspecific sequence variation was found in some species, but T. tonsurans, T. equinum, T. concentricum, T. verrucosum, T. rubrum, T. violaceum, T. eriotrephon, E. floccosum, M. canis, M. ferrugineum, and M. audouinii were invariant. The sequences were found to be relatively conserved among different strains of the same species. The species with the closest resemblance were Arthroderma benhamiae and T. concentricum and T. tonsurans and T. equinum with 100% and 99.8% identity, respectively; the most distant species were M. persicolor and M. amazonicum. The dendrogram obtained from BT2 topology was almost compatible with the species concept based on ITS sequencing, and similar clades and species were distinguished in the BT2 tree. Here, beta tubulin was characterized in a wide range of dermatophytes in order to assess intra- and interspecies variation and resolution and was found to be a taxonomically valuable gene.

Demonstration of Coccidioides immitis and Coccidioides posadasii DNA in soil samples collected from Dinosaur National Monument, Utah.

Abstract: Soil samples were collected in 2006 from Dinosaur National Monument (DNM), Utah, the site of an outbreak of coccidioidomycosis in 2001. DNA was isolated from two soil samples, and polymerase chain reaction (PCR) amplified Coccidioides DNA present in both samples. Ribosomal RNA genes and internal transcribed spacer (ITS) region PCR products were sequenced. Single-nucleotide polymorphisms indicated that the DNA from sample SS06RH was that of Coccidioides immitis, while the DNA from sample SS06UM was C. posadasii. This is the first report to directly demonstrate Coccidioides in soils from DNM and the first to report the presence of both C. immitis and C. posadasii in the same geographic location.

GliA in Aspergillus fumigatus is required for its tolerance to gliotoxin and affects the amount of extracellular and intracellular gliotoxin.

Abstract: Gliotoxin is an important virulence factor of Aspergillus fumigatus. Although GliA putatively belongs to the major facilitator superfamily in the gliotoxin biosynthesis cluster, its roles remain unclear. To determine the function of GliA, we disrupted gliA in A. fumigatus. gliA disruption increased the susceptibility of A. fumigatus to gliotoxin. The gliT and gliA double-disrupted mutant had even higher susceptibility to gliotoxin than each individual disruptant. The extracellular release of gliotoxin was greatly decreased in the gliA disruptant. Mice infected with the gliA disruptant of A. fumigatus showed higher survival rates than those infected with the parent strain. These results strongly indicate that GliA, in addition to GliT, plays a significant role in the tolerance to gliotoxin and protection from extracellular gliotoxin in A. fumigatus by exporting the toxin. This also allows the fungus to evade the harmful effect of its own gliotoxin production. Moreover, GliA contributes to the virulence of A. fumigatus through gliotoxin secretion.

Potential role of Candida albicans germ tube antibody in the diagnosis of deep-seated candidemia.

Abstract: Patients with candidemia may have transient or catheter-related infections without involvement of deep tissues or deep-seated candidiasis. Clinical differentiation of these entities may not be evident with conventional microbiological and imaging methods. Our aim was to determine if the detection of Candida albicans germ tube-specific antibody (CAGTA) in patients with candidemia was related to the extent of the disease. This study was conducted from 2003 to 2012 with 50 patients diagnosed as having candidemia, that is, 29 with deep-seated candidiasis and 21 with non-deep-seated candidiasis. The most common species recovered from samples obtained from these patients were C. albicans, 40%; C. tropicalis, 20%; C. parapsilosis, 18%; and C. glabrata, 12%. Serum samples were processed according to the manufacturer's recommendations (Vircell Microbiologist S.L., Granada, Spain). The CAGTA tests were positive in 1/21 non-deep-seated candidemias (DSCs; 4.76%) and 20/29 DSCs (68.96%; P < 0.01). Accordingly, the values for specificity and positive predictive values of CAGTA for identifying DSC were 95%. We concluded that the presence of a positive CAGTA test in a sample from a patient with candidemia suggests deep-seated candidiasis. Extension screening studies should be considered and origins other than catheters should be searched. Prospective studies are needed to determine the clinical implications of this finding and its potential use in defining the optimal duration of therapy.

Automation of serum (1→3)-beta-D-glucan testing allows reliable and rapid discrimination of patients with and without candidemia.

Abstract: Testing for (1→3)-beta-D-glucan (BDG) is used for detection of invasive fungal infection. However, current assays lack automation and the ability to conduct rapid single-sample testing. The Fungitell assay was adopted for automation and evaluated using clinical samples from patients with culture-proven candidemia and from culture-negative controls in duplicate. A comparison with the standard assay protocol was made in order to establish analytical specifications. With the automated protocol, the analytical measuring range was 8-2500 pg/ml of BDG, and precision testing resulted in coefficients of variation that ranged from 3.0% to 5.5%. Samples from 15 patients with culture-proven candidemia and 94 culture-negative samples were evaluated. All culture-proven samples showed BDG values >80 pg/ml (mean 1247 pg/ml; range, 116-2990 pg/ml), which were considered positive. Of the 94 culture-negative samples, 92 had BDG values <60 pg/ml (mean, 28 pg/ml), which were considered to be negative, and 2 samples were false-positive (≥80 pg/ml; up to 124 pg/ml). Results could be obtained within 45 min and showed excellent agreement with results obtained with the standard assay protocol. The automated Fungitell assay proved to be reliable and rapid for diagnosis of candidemia. It was demonstrated to be feasible and cost efficient for both single-sample and large-scale testing of serum BDG. Its 1-h time-to-result will allow better support for clinicians in the management of antifungal therapy.

Cutaneous mucormycosis and motor vehicle accidents: Findings from an Australian case series

Abstract: Cutaneous disease is the third most frequent manifestation of mucormycosis. The clinical manifestations of and subsequent mortality due to cutaneous mucormycosis are dependent on the mode of acquisition and the host immune status. Here, we describe the epidemiology, clinical presentation, microbiology, and outcomes of 16 cutaneous mucormycosis infections managed in an Australian tertiary hospital over a 15-year period. The proportion with localized (56%), deep (38%), and disseminated (6%) cutaneous disease as well as the overall mortality (25%) were consistent with findings reported in the published literature. Two novel forms of hospital-acquired infection were reported following a sacral pressure sore and insertion of a foreign body during a bone graft procedure. The majority of patients were immunocompetent (75%) and/or suffered trauma (56%) with associated environmental contamination. A novel finding was that motor vehicle accidents (MVAs) accounted for 78% of all trauma-related cases, suggesting MVAs should receive greater recognition as a potential precipitant of cutaneous mucormycosis. Aggressive decontamination and debridement of devitalized tissue following trauma is therefore likely to play an important role in the prevention of this rare but potentially devastating infection.

IFN-γ impairs Trichophyton rubrum proliferation in a murine model of dermatophytosis through the production of IL-1β and reactive oxygen species

Abstract: Trichophyton rubrum is the main etiological agent of dermatophytosis, an infection of the skin that affects millions of people worldwide. In this study, we developed a murine model of the dermatophytosis caused by T. rubrum in which C57BL/6 wild-type, interleukin (IL)-12(-/-), and interferon-gamma (IFN-γ(-/-)) mice were inoculated with 1 × 10(6) conidia/animal. The fungal burden, myeloperoxidase and N-acetylglucosaminidase activities, cytokine and chemokine profiles, and histopathology of the skin were evaluated on the seventh and fourteenth days post infection. Phagocytic indices, intracellular proliferation rates, and oxidative bursts generated by macrophages from WT and IFN-γ(-/-) mice were determined. On day 7 post infection, higher fungal burdens were observed comparison with burdens on day 14 post infection. The IL-12(-/-) and IFN-γ(-/-) mice showed higher fungal burdens on the skin and lower levels of IL-1β. Conversely, the WT mice showed lower fungal burdens with higher production of TNF-α, IL-1β, and chemokine ligand 1/keratinocyte chemoattractant (CXCL1/KC). The macrophages from WT mice proved to be more efficient at engulfing and killing T. rubrum conidia through the production of reactive oxygen species. The results show that our model is a useful tool for understanding the pathogenesis of dermatophytosis caused by T. rubrum and that IL-12 and IFN-γ are pivotal in controlling the infection through the recruitment and activation of neutrophils and macrophages.

Prevalence of Candida albicans-closely related yeasts, Candida africana and Candida dubliniensis, in vulvovaginal candidiasis.

Abstract: Isolates of Candida africana and C. dubliniensis were recovered from patients with vulvovaginal candidiasis (VVC). The isolates were initially identified as C. albicans through use of the API Candida System. We retrospectively reexamined 1014 vaginal isolates presumptively determined to be C. albicans at the Department of Obstetrics and Gynecology of Peking University Shenzhen Hospital from 1 January 2003 through 31 December 2012. Our objective was to determine, via detection of the HWP1 gene, if any of the isolates were C. africana or C. dubliniensis. One and a half percent of these isolates (15/1014) were found to be C. africana, whereas C. dubliniensis was not detected. The 15 C. africana isolates were susceptible to nystatin, fluconazole, itraconazole, miconazole, and clotrimazole. Candida africana could not be recovered from clinical vaginal specimens from the 15 patients at follow-up on days 7-14 and days 30-35 when treated with different antifungal agents. We conclude that C. africana, but not C. dubliniensis, was present in the vaginal samples of patients with VVC. The C. africana isolates were susceptible to the tested antifungal agents. VVC caused by C. africana appears to respond well to current therapies.

An invertebrate model to evaluate virulence in Aspergillus fumigatus: the role of azole resistance.

Abstract: The impact of different mutations in the Aspergillus fumigatus ergosterol biosynthesis pathway on pathogenesis has been evaluated using a simple invertebrate mini host, the caterpillar Galleria mellonella. A set of strains that includes clinical isolates and isogenic mutants with mutations at the cyp51A gene conferring azole resistance were studied. All strains demonstrated a similar in vitro growth pattern and are equally virulent against the insect larvae. These results suggest that in A. fumigatus acquisition of this particular azole-resistance mechanism would not imply any significant change in virulence. G. mellonella may provide a convenient and inexpensive model for the in vivo prescreening of mutants of A. fumigatus, contributing to the generation of a hypotheses that can be further tested in refined experiments in mammalian models.

Molecular identification, genotyping, and antifungal susceptibility testing of clinically relevant Trichosporon species from Argentina

Abstract: Trichosporon species are emerging causative agents of mycoses; most are documented in immunocompromised patients. Species identification is important for epidemiological purposes in order to better define species clinical associations and to improve antifungal treatment. Here, we studied a collection of 41 Trichosporon strains recovered from hospitalized patients in Argentina. All strains were identified by sequencing the D1/D2 domain of 26S, internal transcribed spacer (ITS) regions, and intergenic spacer 1 (IGS1) region. In addition, we determined the IGS1 region genotypes of the suspected T. asahii strains. Antifungal susceptibility of all strains was investigated. Thirty-eight of the 41 strains in this study were identified as follows: 29 T. asahii ,3 T. inkin ,3 T. montevideense ,2 T. faecale, and 1 T. dermatis. The identity of the three remaining strains could not be confirmed. Strain DMic 114126 (Culture collection of the Mycology Department (DMic), National

Identification and typing of the Candida parapsilosis complex: MALDI-TOF MS vs. AFLP.

Abstract: In this study we compare the capability of amplification fragment-length polymorphism (AFLP) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify and subtype isolates of members of the Candida parapsilosis complex (C. parapsilosis, C. orthopsilosis, C. metapsilosis) and Lodderomyces elongisporus, which cannot be differentiated with biochemical methods. Both techniques correctly identified all isolates included in this study and clustered isolates within the different species. DNA-based and mass spectrum-based dendrograms yielded similar outcomes with regard to phylogenetic distance within C. orthopsilosis and C. parapsilosis species. However, a different clustering was obtained for C. metapsilosis for which AFLP was highly effective in differentiating. While MALDI-TOF MS was found to be a reliable method for species-level identification, further studies are required to assess its value as a fungal typing tool.

Exserohilum infections: Review of 48 cases before the 2012 United States outbreak

Abstract: Exserohilum species are soilborne fungi that have been uncommon causes of human disease. The ongoing outbreak in the United States warrants improved understanding of this pathogen. We systematically reviewed all cases of Exserohilum spp. infections published before the outbreak in 2012 in order to provide a better understanding of the organism and its wider spectrum of human disease. Cases of Exserohilum infections were retrieved by searching PubMed. Demographic data, underlying conditions, microbiology, clinical manifestations, therapy, and outcome were recorded and analyzed. Forty-eight evaluable cases were identified from 1975 to 2012. The number of reported cases increased more than twofold during the study period (P < 0.01). Most cases occurred in the southern United States, India, and Israel. Median age of patients was 25 years, with a male predominance. Most infections were due to E. rostratum (60.4%), followed by E. longirostratum (6.3%) and E. mcginnisii (2%), while 31.3% were unidentified species. The most frequent underlying conditions were immunosuppression (27.2%), trauma (16.6%), and atopy (12.5%). Exserohilum disease manifested as systemic (73%), cutaneous (25%), corneal (16.7%), and subcutaneous (10.4%) infection. Antifungal therapy consisted mainly of amphotericin B (44%) alone or combined with a triazole. Surgery was used in 48% of cases and was combined with antifungal therapy in 31%. The all-cause mortality was 23%, which was higher in patients with preexisting immunosuppression (56.2%; odds ratio 15.4; 95% confidence interval, 2.7-88.6). This review of the pre-outbreak reported cases highlights several aspects of epidemiology, clinical presentation, risk factors, and management of this unusual pathogen.

Itraconazole vs. trimethoprim–sulfamethoxazole: a comparative cohort study of 200 patients with paracoccidioidomycosis

Abstract: Paracoccidioidomycosis (PCM) is a systemic mycosis endemic to Latin America. Brazil accounts for approximately 80% of cases, where it represents a major public health issue due to its disabling impact and the number of premature deaths it causes. We present a retrospective cohort study that was conducted in order to better understand factors that relate to cure of the infection in the treatment of 200 patients with PCM. We evaluated the influence of sociodemographic and clinical factors as well as therapeutic regimen (trimethoprim-sulfamethoxazole [TMP-SMX] and itraconazole) on the progress of PCM (cure and noncure). There was a higher incidence of cure (83%) among patients who regularly received treatment for their infections and completed the treatment protocol. Moreover, itraconazole (86.4%) was significantly superior to TMP-SMX (51.3%) in terms of cure rate and had a median treatment period that was significantly shorter (12 months) than that for TMP-SMX (23 months). A Cox proportional hazard regression model showed that use of itraconazole increased the hazard of cure, regardless of sex, age, education, clinical form, completion of treatment, and regularity. Although the results of this study show that itraconazole was the best treatment option for PCM patients, a double-blind, randomized, controlled trial is necessary to confirm this conclusion.

Aspergillus pragensis sp. nov. discovered during molecular reidentification of clinical isolates belonging to Aspergillus section Candidi.

Abstract: The identity of nine clinical isolates recovered from Czech patients and presumptively identified as Aspergillus sp. section Candidi based on colony morphology was revised using sequences of β-tubulin, calmodulin gene sequence, and internal transcribed spacer rDNA. Six isolates were from suspected and proven onychomycosis, one from otitis externa, and two associated with probable invasive aspergillosis. The results showed that one Aspergillus candidus isolate was the cause of otitis externa, and both isolates obtained from sputa of patients with probable invasive aspergillosis were reidentified as A. carneus (sect. Terrei )a ndA. flavus (sect. Flavi). Three isolates from nail scrapings were identified as A. tritici, a verified agent of nondermatophyte onychomycosis. One isolate from toenail was determined to be A. candidus and the two isolates belonged to a hitherto undescribed species, Aspergillus pragensis sp. nov. This species is well supported by phylogenetic analysis based on β-tubulin and calmodulin gene and is distinguishable

Inhaled corticosteroids and Aspergillus fumigatus isolation in cystic fibrosis

Abstract: Aspergillus fumigatus isolation in cultures from respiratory specimens of patients with cystic fibrosis (CF) is quite common; however, the role of A. fumigatus as a pathogen and whether its presence is associated with progression of pulmonary disease remain unclear. We investigated the association between inhaled corticosteroids and the recovery of A. fumigatus by performing a retrospective cohort study of CF patients born between 1988 and 1996. The patients' medical records from their first visit to the CF Center until December 2010 were reviewed. Outcomes were the occurrence of A. fumigatus first isolation, chronic colonization, or the last visit at the CF Center. A number of possible confounders were included in the multivariate logistic regression analysis in order to identify an independent association between inhaled corticosteroids and colonization status. A total of 121 patients were included in the study. Thirty-nine patients (32.2%) had at least one positive culture and 14 (11.6%) developed chronic colonization. Multivariate logistic regression analysis was used to determine the independent effect of inhaled corticosteroids on the odds of first isolation (odds ratio [OR], 1.165; 95% confidence interval [CI], 1.015-1.337; P = 0.029) and chronic colonization (OR, 1.180; 95% CI, 1.029-1.353; P = 0.018). In conclusion, A. fumigatus first isolation and chronic colonization are associated with the duration of inhaled corticosteroid treatment.

In vitro inhibitory effect of miltefosine against strains of Histoplasma capsulatum var. capsulatum and Sporothrix spp.

Abstract: Miltefosine (MIL), originally developed for use in cancer chemotherapy, has been shown to have important antifungal activity against several pathogenic fungi. Our aim in this study was to determine the in vitro activity of MIL against the dimorphic fungi Histoplasma capsulatum and Sporothrix spp. This was done using the broth microdilution method. MIL had an in vitro inhibitory effect against all strains of H. capsulatum var. capsulatum and Sporothrix spp. analyzed. The minimal inhibitory concentrations (MIC) varied from 0.25µg/ml to 2µg/ml for H. capsulatum var. capsulatum in the filamentous phase and from 0.125µg/ml to 1µg/ml in the yeast phase. The MIC interval for Sporothrix spp. in the filamentous phase was 0.25‐2µg/ml. The minimal fungicidal concentrations (MFCs) were ≤4µg/ml for isolates of both analyzed species. This study demonstrates that MIL has an antifungal effect in vitro against two potentially pathogenic fungi and that more studies should be performed in order to evaluate its applicability in vivo.

Melanocytes and melanin represent a first line of innate immunity against Candida albicans.

Abstract: Melanocytes are dendritic cells located in the skin and mucosae that synthesize melanin. Some infections induce hypo- or hyperpigmentation, which is associated with the activation of Toll-like receptors (TLRs), especially TLR4. Candida albicans is an opportunist pathogen that can switch between blastoconidia and hyphae forms; the latter is associated with invasion. Our objectives in this study were to ascertain whether C. albicans induces pigmentation in melanocytes and whether this process is dependent on TLR activation, as well as relating this with the antifungal activity of melanin as a first line of innate immunity against fungal infections. Normal human melanocytes were stimulated with C. albicans supernatants or with crude extracts of the blastoconidia or hyphae forms, and pigmentation and TLR2/TLR4 expression were measured. Expression of the melanosomal antigens Melan-A and gp100 was examined for any correlation with increased melanin levels or antifungal activity in melanocyte lysates. Melanosomal antigens were induced earlier than cell pigmentation, and hyphae induced stronger melanization than blastoconidia. Notably, when melanocytes were stimulated with crude extracts of C. albicans, the cell surface expression of TLR2/TLR4 began at 48 h post-stimulation and peaked at 72 h. At this time, blastoconidia induced both TLR2 and TLR4 expression, whereas hyphae only induced TLR4 expression. Taken together, these results suggest that melanocytes play a key role in innate immune responses against C. albicans infections by recognizing pathogenic forms of C. albicans via TLR4, resulting in increased melanin content and inhibition of infection.

Prevalence, persistence, and phenotypic variation of Aspergillus fumigatus in the outdoor environment in Manchester, UK, over a 2-year period

Abstract: Aspergillus fumigatus, an opportunistic fungal pathogen that causes invasive aspergillosis in immunosuppressed patients, is considered to be the world’s most dangerous mould. It is widely distributed in the environment, and airborne asexual conidia serve as the main mode of transport for pulmonary lung infection. It is important to monitor seasonal airborne conidia levels when assessing the risk of acquiring this infection. In this study, air was sampled for total viable fungal spores and viable A. fumigatus conidia monthly over a 2-year period (2009 and 2010) close to Manchester, UK, city center. Total viable airborne fungal counts varied seasonally, peaking in the summer and autumn for both years and reaching levels of approximately 1100– 1400 colony-forming units (CFU)/m 3 ; counts were strongly positively correlated to mean temperature (R 2 = 0.697). By contrast, A. fumigatus viable airborne counts were not seasonally associated; persistent low levels were between 3 and 20 CFU/m 3 and were not correlated with mean temperature (R 2 = 0.018). A total of 220 isolates of A. fumigatus were recovered on potato dextrose agar (PDA) at 45 ◦ C, and internal transcribed spacer sequencing and restriction digestion of a partial polymerase chain reaction amplicon of the β-tubulin gene (benA) of 34 randomly selected isolates were used to confirm the isolates as A. fumigatus. When the colony radial growth rates (Kr) were determined, the highest rates were observed on PDA, followed by Vogel’s medium supplemented with phosphatidylcholine and Vogel’s medium alone. Clinical isolates had a significantly higher mean colony Kr on PDA compared with environmental isolates.

A family of glutathione peroxidases contributes to oxidative stress resistance in Candida albicans.

Abstract: Candida albicans is a well-adapted human commensal but is also a facultative pathogen that can cause superficial and systemic infections. Its remarkable capacity to thrive within the human host relies on its ability to adapt and respond to the local environment of different niches. C. albicans is able to cope with oxidative stress in a coordinated fashion via upregulation of different protective mechanisms. Here, we unravel the role of a family of glutathione peroxidase (GPx), designated Gpx31, Gpx32, and Gpx33, in oxidative stress resistance. We show that GPx activity in C. albicans is induced upon exposure to peroxides and that this enzymatic activity is required for full resistance to oxidative stress. The GPx activity relies on the presence of GPX31, with no apparent contribution from GPX32 and GPX33 during in vitro short-term (3 h) exposure to peroxides. However, a triple gpx31-33Δ/Δ mutant exhibited a more pronounced sensitivity than a single gpx31Δ/Δ mutant on solid media in the presence of oxidants, suggesting that GPX32 and GPX33 may be involved in long-term adaptation to oxidative stress. Interestingly, reintegration of a single allele of GPX31 was sufficient to restore the wild-type phenotype in both the single and triple mutants. We found that mutants lacking GPX31-33 were more susceptible to killing by phagocytic cells, suggesting that GPxs are required for full resistance to innate immune effector cells. Despite the sensitivity to oxidative stress and phagocytes, these mutants were not affected in their virulence in the chicken embryo model of candidiasis.

Synergistic effect of amphotericin B and tyrosol on biofilm formed by Candida krusei and Candida tropicalis from intrauterine device users.

Abstract: The presence of intrauterine contraceptive devices (IUDs) provides a solid surface for attachment of microorganisms and an ideal niche for the biofilm to form and flourish. Vaginal candidiasis is often associated with the use of IUDs. Treatment of vaginal candidiasis that develops in connection with IUD use requires their immediate removal. Here we present in vitro evidence to support the use of combination therapy to inhibit Candida biofilm. Twenty-three clinical Candida isolates (10 C. krusei and 13 C. tropicalis) recovered from endocervical swabs obtained from IUD and non-IUD users were assessed for biofilm-formation ability. The rate of isolation of Candida did not differ significantly among IUD and non-IUD users (P = 0.183) but the biofilm-formation ability of isolates differed significantly (P = 0.02). An in vitro biofilm model with the obtained isolates was subjected to treatment with amphotericin B tyrosol and a combination of amphotericin B and tyrosol. Inhibition of biofilm by amphotericin B or tyrosol was found to be concentration dependent with 50% reduction (P < 0.05) at 4 mg/l and 80 muM respectively. Hence a combination effect of tyrosol and amphotericin B was studied. Interestingly approximately 90% reduction in biofilm was observed with use of 80 muM tyrosol combined with 4 mg/l amphotericin B (P < 0.001). This represents a first step in establishing an appropriate antibiofilm therapy when yeasts are present. (c) The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions please e-mail: journals.permissions@oup.com.

Genomics in Coccidioides: Insights into evolution, ecology, and pathogenesis

Abstract: Coccidioides immitis and C. posadasii, the causative agents of the mammalian disease coccidioidomycosis, are dimorphic fungal pathogens distributed throughout desert-like environments in North and South America. Coccidioides spp. are members of the Onygenales, a diverse group of pathogenic and nonpathogenic fungi. Recently, full genomes have been published for Coccidioides and a number of other Onygenales species. Phylogenomic comparisons and additional studies in Coccidioides population genomics and gene expression have shed light on the ecology and pathogenesis of Coccidioides and the other medically important species in this clade. Observed patterns of gene family expansion/contraction and evidence of gene flow have provided insight to the evolution of Coccidioides and greatly broadened our understanding of the diversity and sources of genetic variation found in fungi. In the future, expansion of the number of sequenced isolates from all populations will allow deeper insight into the evolutionary processes that have shaped this unique human pathogen. In addition, deep sequencing of isolates from a single Coccidioides population and pairing of those data with phenotype information on growth and pathogenicity for genome-wide association analysis will allow researchers to find genes responsible for any phenotype, virulence included, that shows variation in the population.