Posaconazole treatment of refractory eumycetoma and chromoblastomycosis

Eumycetoma and chromoblastomycosis are chronic, disfiguring fungal infections of the subcutaneous tissue that rarely resolve spontaneously. Most patients do not achieve sustained long-term benefits from available treatments; therefore, new therapeutic options are needed. We evaluated the efficacy of posaconazole, a new extended-spectrum triazole antifungal agent, in 12 patients with eumycetoma or chromoblastomycosis refractory to existing antifungal therapies. Posaconazole 800 mg/d was given in divided doses for a maximum of 34 months. Complete or partial clinical response was considered a success; stable disease or failure was considered a nonsuccess. All 12 patients had proven infections refractory to standard therapy. Clinical success was reported for five of six patients with eumycetoma and five of six patients with chromoblastomycosis. Two patients were reported to have stable disease. As part of a treatment-use extension protocol, two patients with eumycetoma who initially had successful outcome were successfully retreated with posaconazole after a treatment hiatus of > 10 months. Posaconazole was well tolerated during long-term administration (up to 1015 d). Posaconazole therapy resulted in successful outcome in most patients with eumycetoma or chromoblastomycosis refractory to standard therapies, suggesting that posaconazole may be an important treatment option for these diseases.

Multiplex ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous quantification in human plasma of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-N-oxide, anidulafungin, and caspofungin.

Therapeutic drug monitoring (TDM) may contribute to optimizing the efficacy and safety of antifungal therapy because of the large variability in drug pharmacokinetics. Rapid, sensitive, and selective laboratory methods are needed for efficient TDM. Quantification of several antifungals in a single analytical run may best fulfill these requirements. We therefore developed a multiplex ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method requiring 100 μl of plasma for simultaneous quantification within 7 min of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-N-oxide, caspofungin, and anidulafungin. Protein precipitation with acetonitrile was used in a single extraction procedure for eight analytes. After reverse-phase chromatographic separation, antifungals were quantified by electrospray ionization-triple-quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. Deuterated isotopic compounds of azole antifungals were used as internal standards. The method was validated based on FDA recommendations, including assessment of extraction yields, matrix effect variability (<9.2%), and analytical recovery (80.1 to 107%). The method is sensitive (lower limits of azole quantification, 0.01 to 0.1 μg/ml; those of echinocandin quantification, 0.06 to 0.1 μg/ml), accurate (intra- and interassay biases of -9.9 to +5% and -4.0 to +8.8%, respectively), and precise (intra- and interassay coefficients of variation of 1.2 to 11.1% and 1.2 to 8.9%, respectively) over clinical concentration ranges (upper limits of quantification, 5 to 50 μg/ml). Thus, we developed a simple, rapid, and robust multiplex UPLC-MS/MS assay for simultaneous quantification of plasma concentrations of six antifungals and two metabolites. This offers, by optimized and cost-effective lab resource utilization, an efficient tool for daily routine TDM aimed at maximizing the real-time efficacy and safety of different recommended single-drug antifungal regimens and combination salvage therapies, as well as a tool for clinical research.

Ultra-performance liquid chromatography mass spectrometry and sensitive bioassay methods for quantification of posaconazole plasma concentrations after oral dosing.

Posaconazole (POS) is a new antifungal agent for prevention and therapy of mycoses in immunocompromised patients. Variable POS pharmacokinetics after oral dosing may influence efficacy: a trough threshold of 0.5 ?g/ml has been recently proposed. Measurement of POS plasma concentrations by complex chromatographic techniques may thus contribute to optimize prevention and management of life-threatening infections. No microbiological analytical method is available. The objective of this study was to develop and validate a new simplified ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method and a sensitive bioassay for quantification of POS over the clinical plasma concentration range. The UPLC-MS/MS equipment consisted of a triple quadrupole mass spectrometer, an electrospray ionization (ESI) source, and a C(18) analytical column. The Candida albicans POS-hypersusceptible mutant (MIC of 0.002 ?g/ml) ?cdr1 ?cdr2 ?flu ?mdr1 ?can constructed by targeted deletion of multidrug efflux transporters and calcineurin genes was used for the bioassay. POS was extracted from plasma by protein precipitation with acetonitrile-methanol (75%/25%, vol/vol). Reproducible standard curves were obtained over the range 0.014 to 12 (UPLC-MS/MS) and 0.028 to 12 ?g/ml (bioassay). Intra- and interrun accuracy levels were 106% ± 2% and 103% ± 4% for UPLC-MS/MS and 102% ± 8% and 104% ± 1% for bioassay, respectively. The intra- and interrun coefficients of variation were 7% ± 4% and 7% ± 3% for UPLC-MS/MS and 5% ± 3% and 4% ± 2% for bioassay, respectively. An excellent correlation between POS plasma concentrations measured by UPLC-MS/MS and bioassay was found (concordance, 0.96). In 26 hemato-oncological patients receiving oral POS, 27/69 (39%) trough plasma concentrations were lower than 0.5 ?g/ml. The UPLC-MS/MS method and sensitive bioassay offer alternative tools for accurate and precise quantification of the plasma concentrations in patients receiving oral posaconazole.

Posaconazole salvage treatment in paediatric patients: a multicentre survey

While a paediatric dosage has not been defined, posaconazole is occasionally being used in children. We conducted a multicentre retrospective survey and identified 15 patients (median age 10 years [range 3.6-17.5]) who received posaconazole salvage therapy for proven (9 patients) or probable (6 patients) invasive fungal infections. Posaconazole was administered for a median of 32 days (range 4-262) at a median dosage of 21 mg/kg (range 4.8-33.3). None of the patients discontinued therapy due to adverse events, which were mostly mild and observed in 11 patients. Complete or partial responses were observed in 4/7 patients with zygomycosis, 3/4 patients with invasive mould infection, 1/2 patients with invasive aspergillosis and 1/2 patients with chronic disseminated candidiasis. We conclude from the data that posaconazole displays favourable safety and tolerance and may be useful for management of individual paediatric patients with invasive infections.

Extensive long-standing chromomycosis due to Fonsecaea pedrosoi: Three cases with relevant improvement under voriconazole therapy

Objective: To evaluate voriconazole in the treatment of extensive cases of chromomycosis. Chromomycosis is a chronic infection, which is extremely difficult to eradicate, and is caused by dematiaceous (dark-colored) fungi which affect the skin and subcutaneous tissues, with Fonsecaea pedrosoi being the major etiologic agent. Drugs such as itraconazole, terbinafine, posaconazole and amphotericin B have been employed with variable results. Methods: We treated three Caucasian male patients (ages 44, 57 and 77 years), two were farmers and one a trash collector, with long-standing (20, 10 and 21 years of disease, respectively) and extensive chromomycosis (one lower limb affected, at least) due to Fonsecaea pedrosoi. All patients had received previous therapy with the formerly indicated drugs itraconazole and terbinafine for several months either without or with incomplete response. After that, we started treatment with voriconazole per os 200 mg twice a day. Results: The patients were treated with voriconazole for 12 months until there was clinical and mycological improvement. Clinical response was evident after 30-50 days. One patient developed visual abnormalities and tremors, and the voriconazole was reduced to 200 mg/day without impairment of the clinical and mycological response. The same patient presented photosensitive dermatitis after 12 months of therapy and the voriconazole was stopped. All patients showed elevations of serum gamma-glutamyl transpeptidase (GGT) during the treatment without clinical relevance. Moreover, our three patients obtained partial response with this therapy. Conclusions: This is the first report with a case series of chromomycosis treated with voriconazole. Despite its high cost, voriconazole is a safe and possibly promising drug for use on extensive chromomycosis refractory to conventional treatment.

Molecular identification and antifungal susceptibility profiles of Candida parapsilosis complex species isolated from culture collection of clinical samples

AbstractINTRODUCTION:Candida parapsilosis is a common yeast species found in cases of onychomycosis and candidemia associated with infected intravascular devices. In this study, we differentiated Candida parapsilosis sensu stricto, Candida orthopsilosis , and Candida metapsilosis from a culture collection containing blood and subungual scraping samples. Furthermore, we assessed the in vitro antifungal susceptibility of these species to fluconazole, itraconazole, voriconazole, posaconazole, amphotericin B, and caspofungin.METHODS:Differentiation of C. parapsilosis complex species was performed by amplification of the secondary alcohol dehydrogenase (SADH) gene and digestion by the restriction enzyme Ban I. All isolates were evaluated for the determination of minimal inhibitory concentrations using Etest, a method for antifungal susceptibility testing.RESULTS:Among the 87 isolates, 78 (89.7%) were identified as C. parapsilosis sensu stricto , five (5.7%) were identified as C. orthopsilosis , and four (4.6%) were identified as C. metapsilosis . Analysis of antifungal susceptibility showed that C. parapsilosis sensu strictoisolates were less susceptible to amphotericin B and itraconazole. One C. parapsilosis sensu stricto isolate was resistant to amphotericin B and itraconazole. Moreover, 10.2% of C. parapsilosis sensu stricto isolates were resistant to caspofungin. Two C. parapsilosis sensu strictoisolates and one C. metapsilosis isolate were susceptible to fluconazole in a dose-dependent manner.CONCLUSIONS:We reported the first molecular identification of C. parapsilosiscomplex species in State of Goiás, Brazil. Additionally, we showed that although the three species exhibited differences in antifungal susceptibility profiles, the primary susceptibility of this species was to caspofungin.

NEW METHODS FOR DETECTION, SUSCEPTIBILITY TESTING AND BIOFILM ERADICATION OF DIFFICULT ORGANISMS

Aujourd'hui, les problèmes des maladies infectieuses concernent l'émergence d'infections difficiles à traiter, telles que les infections associées aux implants et les infections fongiques invasives chez les patients immunodéprimés. L'objectif de cette thèse était de développer des stratégies pour l'éradication des biofilms bactériens (partie 1), ainsi que d'étudier des méthodes innovantes pour la détection microbienne, pour l'établissement de nouveaux tests de sensibilité (partie 2). Le traitement des infections associées aux implants est difficile car les biofilms bactériens peuvent résister à des niveaux élevés d'antibiotiques. A ce jour, il n'y a pas de traitement optimal défini contre des infections causées par des bactéries de prévalence moindre telles que Enterococcus faecalis ou Propionibacterium acnés. Dans un premier temps, nous avons démontré une excellente activité in vitro de la gentamicine sur une souche de E. faecalis en phase stationnaire de croissance Nous avons ensuite confirmé l'activité de la gentamicine sur un biofilm précoce en modèle expérimental animal à corps étranger avec un taux de guérison de 50%. De plus, les courbes de bactéricidie ainsi que les résultats de calorimétrie ont prouvé que l'ajout de gentamicine améliorait l'activité in vitro de la daptomycine, ainsi que celle de la vancomycine. In vivo, le schéma thérapeutique le plus efficace était l'association daptomycine/gentamicine avec un taux de guérison de 55%. En établissant une nouvelle méthode pour l'évaluation de l'activité des antimicrobiens vis-à-vis de micro-organismes en biofilm, nous avons démontré que le meilleur antibiotique actif sur les biofilms à P. acnés était la rifampicine, suivi par la penicilline G, la daptomycine et la ceftriaxone. Les études conduites en modèle expérimental animal ont confirmé l'activité de la rifampicine seule avec un taux de guérison 36%. Le meilleur schéma thérapeutique était au final l'association rifampicine/daptomycine avec un taux de guérison 63%. Les associations de rifampicine avec la vancomycine ou la levofloxacine présentaient des taux de guérisons respectivement de 46% et 25%. Nous avons ensuite étudié l'émergence in vitro de la résistance à la rifampicine chez P. acnés. Nous avons observé un taux de mutations de 10"9. La caractérisation moléculaire de la résistance chez les mutant-résistants a mis en évidence l'implication de 5 mutations ponctuelles dans les domaines I et II du gène rpoB. Ce type de mutations a déjà été décrit au préalable chez d'autres espèces bactériennes, corroborant ainsi la validité de nos résultats. La deuxième partie de cette thèse décrit une nouvelle méthode d'évaluation de l'efficacité des antifongiques basée sur des mesures de microcalorimétrie isotherme. En utilisant un microcalorimètre, la chaleur produite par la croissance microbienne peut être-mesurée en temps réel, très précisément. Nous avons évalué l'activité de l'amphotéricine B, des triazolés et des échinocandines sur différentes souches de Aspergillus spp. par microcalorimétrie. La présence d'amphotéricine Β ou de triazole retardait la production de chaleur de manière concentration-dépendante. En revanche, pour les échinochandines, seule une diminution le pic de « flux de chaleur » a été observé. La concordance entre la concentration minimale inhibitrice de chaleur (CMIC) et la CMI ou CEM (définie par CLSI M38A), avec une marge de 2 dilutions, était de 90% pour l'amphotéricine B, 100% pour le voriconazole, 90% pour le pozoconazole et 70% pour la caspofongine. La méthode a été utilisée pour définir la sensibilité aux antifongiques pour d'autres types de champignons filamenteux. Par détermination microcalorimétrique, l'amphotéricine B s'est avéré être l'agent le plus actif contre les Mucorales et les Fusarium spp.. et le voriconazole le plus actif contre les Scedosporium spp. Finalement, nous avons évalué l'activité d'associations d'antifongiques vis-à-vis de Aspergillus spp. Une meilleure activité antifongique était retrouvée avec l'amphotéricine B ou le voriconazole lorsque ces derniers étaient associés aux échinocandines vis-à-vis de A. fumigatus. L'association échinocandine/amphotéricine B a démontré une activité antifongique synergique vis-à-vis de A. terreus, contrairement à l'association échinocandine/voriconazole qui ne démontrait aucune amélioration significative de l'activité antifongique. - The diagnosis and treatment of infectious diseases are today increasingly challenged by the emergence of difficult-to-manage situations, such as infections associated with medical devices and invasive fungal infections, especially in immunocompromised patients. The aim of this thesis was to address these challenges by developing new strategies for eradication of biofilms of difficult-to-treat microorganisms (treatment, part 1) and investigating innovative methods for microbial detection and antimicrobial susceptibility testing (diagnosis, part 2). The first part of the thesis investigates antimicrobial treatment strategies for infections caused by two less investigated microorganisms, Enterococcus faecalis and Propionibacterium acnes, which are important pathogens causing implant-associated infections. The treatment of implant-associated infections is difficult in general due to reduced susceptibility of bacteria when present in biofilms. We demonstrated an excellent in vitro activity of gentamicin against E. faecalis in stationary growth- phase and were able to confirm the activity against "young" biofilms (3 hours) in an experimental foreign-body infection model (cure rate 50%). The addition of gentamicin improved the activity of daptomycin and vancomycin in vitro, as determined by time-kill curves and microcalorimetry. In vivo, the most efficient combination regimen was daptomycin plus gentamicin (cure rate 55%). Despite a short duration of infection, the cure rates were low, highlighting that enterococcal biofilms remain difficult to treat despite administration of newer antibiotics, such as daptomycin. By establishing a novel in vitro assay for evaluation of anti-biofilm activity (microcalorimetry), we demonstrated that rifampin was the most active antimicrobial against P. acnes biofilms, followed by penicillin G, daptomycin and ceftriaxone. In animal studies we confirmed the anti-biofilm activity of rifampin (cure rate 36% when administered alone), as well as in combination with daptomycin (cure rate 63%), whereas in combination with vancomycin or levofloxacin it showed lower cure rates (46% and 25%, respectively). We further investigated the emergence of rifampin resistance in P. acnes in vitro. Rifampin resistance progressively emerged during exposure to rifampin, if the bacterial concentration was high (108 cfu/ml) with a mutation rate of 10"9. In resistant isolates, five point mutations of the rpoB gene were found in cluster I and II, as previously described for staphylococci and other bacterial species. The second part of the thesis describes a novel real-time method for evaluation of antifungals against molds, based on measurements of the growth-related heat production by isothermal microcalorimetry. Current methods for evaluation of antifungal agents against molds, have several limitations, especially when combinations of antifungals are investigated. We evaluated the activity of amphotericin B, triazoles (voriconazole, posaconazole) and echinocandins (caspofungin and anidulafungin) against Aspergillus spp. by microcalorimetry. The presence of amphotericin Β or a triazole delayed the heat production in a concentration-dependent manner and the minimal heat inhibition concentration (MHIC) was determined as the lowest concentration inhibiting 50% of the heat produced at 48 h. Due to the different mechanism of action echinocandins, the MHIC for this antifungal class was determined as the lowest concentration lowering the heat-flow peak with 50%. Agreement within two 2-fold dilutions between MHIC and MIC or MEC (determined by CLSI M38A) was 90% for amphotericin B, 100% for voriconazole, 90% for posaconazole and 70% for caspofungin. We further evaluated our assay for antifungal susceptibility testing of non-Aspergillus molds. As determined by microcalorimetry, amphotericin Β was the most active agent against Mucorales and Fusarium spp., whereas voriconazole was the most active agent against Scedosporium spp. Finally, we evaluated the activity of antifungal combinations against Aspergillus spp. Against A. jumigatus, an improved activity of amphotericin Β and voriconazole was observed when combined with an echinocandin. Against A. terreus, an echinocandin showed a synergistic activity with amphotericin B, whereas in combination with voriconazole, no considerable improved activity was observed.

ESCMID* guideline for the diagnosis and management of Candida diseases 2012: prevention and management of invasive infections in neonates and children caused by Candida spp.

Invasive candidiasis (IC) is a relatively common syndrome in neonates and children and is associated with significant morbidity and mortality. These guidelines provide recommendations for the prevention and treatment of IC in neonates and children. Appropriate agents for the prevention of IC in neonates at high risk include fluconazole (A-I), nystatin (B-II) or lactoferrin ± Lactobacillus (B-II). The treatment of IC in neonates is complicated by the high likelihood of disseminated disease, including the possibility of infection within the central nervous system. Amphotericin B deoxycholate (B-II), liposomal amphotericin B (B-II), amphotericin B lipid complex (ABLC) (C-II), fluconazole (B-II), micafungin (B-II) and caspofungin (C-II) can all be potentially used. Recommendations for the prevention of IC in children are largely extrapolated from studies performed in adults with concomitant pharmacokinetic data and models in children. For allogeneic HSCT recipients, fluconazole (A-I), voriconazole (A-I), micafungin (A-I), itraconazole (B-II) and posaconazole (B-II) can all be used. Similar recommendations are made for the prevention of IC in children in other risk groups. With several exceptions, recommendations for the treatment of IC in children are extrapolated from adult studies, with concomitant pharmacokinetic studies. Amphotericin B deoxycholate (C-I), liposomal amphotericin B (A-I), ABLC (B-II), micafungin (A-I), caspofungin (A-I), anidulafungin (B-II), fluconazole (B-I) and voriconazole (B-I) can all be used.

Real-time antifungal susceptibility testing of Mucor spp., Fusarium spp. and Scedosporium spp. by isothermal microcalorimetry

Objective: Aspergillus species are the main pathogens causing invasive fungal infections but the prevalence of other mould species is rising. Resistance to antifungals among these new emerging pathogens presents a challenge for managing of infections. Conventional susceptibility testing of non-Aspergillus species is laborious and often difficult to interpret. We evaluated a new method for real-time susceptibility testing of moulds based on their of growth-related heat production.Methods: Laboratory and clinical strains of Mucor spp. (n = 4), Scedoporium spp. (n = 4) and Fusarium spp. (n = 5) were used. Conventional MIC was determined by microbroth dilution. Isothermal microcalorimetry was performed at 37 C using Sabouraud dextrose broth (SDB) inoculated with 104 spores/ml (determined by microscopical enumeration). SDB without antifungals was used for evaluation of growth characteristics. Detection time was defined as heat flow exceeding 10 lW. For susceptibility testing serial dilutions of amphotericin B, voriconazole, posaconazole and caspofungin were used. The minimal heat inhibitory concentration (MHIC) was defined as the lowest antifungal concentration, inhbiting 50% of the heat produced by the growth control at 48 h or at 24 h for Mucor spp. Susceptibility tests were performed in duplicate.Results: Tested mould genera had distinctive heat flow profiles with a median detection time (range) of 3.4 h (1.9-4.1 h) for Mucor spp, 11.0 h (7.1-13.7 h) for Fusarium spp and 29.3 h (27.4-33.0 h) for Scedosporium spp. Graph shows heat flow (in duplicate) of one representative strain from each genus (dashed line marks detection limit). Species belonging to the same genus showed similar heat production profiles. Table shows MHIC and MIC ranges for tested moulds and antifungals.Conclusions: Microcalorimetry allowed rapid detection of growth of slow-growing species, such as Fusarium spp. and Scedosporium spp. Moreover, microcalorimetry offers a new approach for antifungal susceptibility testing of moulds, correlating with conventional MIC values. Interpretation of calorimetric susceptibility data is easy and real-time data on the effect of different antifungals on the growth of the moulds is additionally obtained. This method may be used for investigation of different mechanisms of action of antifungals, new substances and drug-drug combinations.

Ergosterol biosynthesis and drug development for Chagas disease

This article presents an overview of the currently available drugs nifurtimox (NFX) and benznidazole (BZN) used against Trypanosoma cruzi, the aetiological agent of Chagas disease; herein we discuss their limitations along with potential alternatives with a focus on ergosterol biosynthesis inhibitors (EBI). These compounds are currently the most advanced candidates for new anti-T. cruzi agents given that they block de novo production of 24-alkyl-sterols, which are essential for parasite survival and cannot be replaced by a host's own cholesterol. Among these compounds, new triazole derivatives that inhibit the parasite's C14± sterol demethylase are the most promising, as they have been shown to have curative activity in murine models of acute and chronic Chagas disease and are active against NFX and BZN-resistant T. cruzi strains; among this class of compounds, posaconazole (Schering-Plough Research Institute) and ravuconazole (Eisai Company) are poised for clinical trials in Chagas disease patients in the short term. Other T. cruzi-specific EBI, with in vitro and in vivo potency, include squalene synthase, lanosterol synthase and squalene epoxidase-inhibitors as well as compounds with dual mechanisms of action (ergosterol biosynthesis inhibition and free radical generation), but they are less advanced in their development process. The main putative advantages of EBI over currently available therapies include their higher potency and selectivity in both acute and chronic infections, activity against NFX and BZN-resistant T. cruzi strains, and much better tolerability and safety profiles. Limitations may include complexity and cost of manufacture of the new compounds. As for any new drug, such compounds will require extensive clinical testing before being introduced for clinical use, and the complexity of such studies, particularly in chronic patients, will be compounded by the current limitations in the verification of true parasitological cures for T. cruzi infections.

Epidemiology and antifungal susceptibility of bloodstream fungal isolates in pediatric patients: a Spanish multicenter prospective survey.

Data on fungemia epidemiology and antifungal susceptibility of isolates from children are scarce, leading frequently to pediatric empirical treatment based on available adult data. The present study was designed to update the epidemiological, mycological, and in vitro susceptibility data on fungal isolates from children with fungemia in Spain. All fungemia episodes were identified prospectively by blood culture over 13 months at 30 hospitals. Tests of susceptibility to amphotericin B, flucytosine, fluconazole, itraconazole, posaconazole, voriconazole, anidulafungin, caspofungin, and micafungin were performed at participant institutions by a microdilution colorimetric method. New species-specific clinical breakpoints for fluconazole, voriconazole, and echinocandins were also applied. A total of 203 episodes of fungemia in 200 children were identified. A higher proportion of fungal isolates was from general wards than intensive care units (ICU). Candida parapsilosis (46.8%), Candida albicans (36.5%), Candida tropicalis (5.9%), Candida glabrata (3.9%), and Candida guilliermondii (2.5%) were the leading species. C. parapsilosis was the predominant species except in neonates. C. albicans was the most frequent in neonatal ICU settings (51.9%). Intravascular catheter (79.3%), surgery (35%), prematurity (30%), and neutropenia (11%) were the most frequent predisposing factors. Most Candida isolates (95.1%) were susceptible to all antifungals. When the new species-specific clinical breakpoints were applied, all C. parapsilosis isolates were susceptible to echinocandins except one, which was micafungin resistant. This is the largest published series of fungemia episodes in the pediatric setting. C. parapsilosis is the most prevalent species in Spain, followed by C. albicans and C. tropicalis. Resistance to azole and echinocandin agents is extremely rare among Candida species. The fluconazole resistance rate in Spain has decreased in the last 10 years.

First description of Candida nivariensis in Brazil: antifungal susceptibility profile and potential virulence attributes

This study evaluated the antifungal susceptibility profile and the production of potential virulence attributes in a clinical strain of Candida nivariensis for the first time in Brazil, as identified by sequencing the internal transcribed spacer (ITS)1-5.8S-ITS2 region and D1/D2 domains of the 28S of the rDNA. For comparative purposes, tests were also performed with reference strains. All strains presented low planktonic minimal inhibitory concentrations (PMICs) to amphotericin B (AMB), caspofungin (CAS), and voriconazole. However, our strain showed elevated planktonic MICs to posaconazole (POS) and itraconazole, in addition to fluconazole resistance. Adherence to inert surfaces was conducted onto glass and polystyrene. The biofilm formation and antifungal susceptibility on biofilm-growing cells were evaluated by crystal violet staining and a XTT reduction assay. All fungal strains were able to bind both tested surfaces and form biofilm, with a binding preference to polystyrene (p < 0.001). AMB promoted significant reductions (≈50%) in biofilm production by our C. nivariensis strain using both methodologies. This reduction was also observed for CAS and POS, but only in the XTT assay. All strains were excellent protease producers and moderate phytase producers, but lipases were not detected. This study reinforces the pathogenic potential of C. nivariensis and its possible resistance profile to the azolic drugs generally used for candidiasis management.

Disseminated Rhizopus microsporus infection cured by salvage allogeneic hematopoietic stem cell transplantation, antifungal combination therapy, and surgical resection.

O. Lebeau, C. Van Delden, J. Garbino, J. Robert, F. Lamoth, J. Passweg, Y. Chalandon. Disseminated Rhizopus microsporus infection cured by salvage allogeneic hematopoietic stem cell transplantation, antifungal combination therapy, and surgical resection. Transpl Infect Dis 2010. All rights reserved Abstract: Invasive Zygomycetes infection complicating prolonged neutropenia is associated with high mortality in the absence of immune recovery. We report a patient who developed disseminated zygomycosis due to Rhizopus microsporus during induction chemotherapy for acute myeloid leukemia. Rescue allogeneic hematopoietic stem cell transplantation (allo-HSCT) was performed as her only chance of cure of this infection and to treat refractory leukemia. Posaconazole combined with liposomal amphotericin B contained the zygomycosis during prolonged neutropenia due to allo-HSCT followed by intense immunosuppression for grade IV acute graft-versus-host disease. Surgical removal of all infected sites after immune recovery, with prolonged posaconazole treatment, ultimately cured the infection. New combination antifungal therapies might sufficiently control disseminated zygomycosis to allow allo-HSCT to be performed, assuring life-saving immune recovery. Surgery appears to be necessary for definite cure of these infections.

Characterization of a new clinical yeast species, Candida tunisiensis sp. nov., isolated from a strain collection from Tunisian hospitals.

From a collection of yeast isolates isolated from patients in Tunisian hospitals between September 2006 and July 2010, the yeast strain JEY63 (CBS 12513), isolated from a 50-year-old male that suffered from oral thrush, could not be identified to the species level using conventional methods used in clinical laboratories. These methods include matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), germ tube formation, and the use of CHROMagar Candida and metabolic galleries. Sequence analysis of the nuclear rRNA (18S rRNA, 5.8S rRNA, and 26S rRNA) and internal transcribed spacer regions (ITS1 and ITS2) indicated that the ribosomal DNA sequences of this species were not yet reported. Multiple gene phylogenic analyses suggested that this isolate clustered at the base of the Dipodascaceae (Saccharomycetales, Saccharomycetes, and Ascomycota). JEY63 was named Candida tunisiensis sp. nov. according to several phenotypic criteria and its geographical origin. C. tunisiensis was able to grow at 42°C and does not form chlamydospores and hyphae but could grow as yeast and pseudohyphal forms. C. tunisiensis exhibited most probably a haploid genome with an estimated size of 10 Mb on at least three chromosomes. Using European Committee for Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) Candida albicans susceptibility breakpoints as a reference, C. tunisiensis was resistant to fluconazole (MIC = 8 μg/ml), voriconazole (MIC = 0.5 μg/ml), itraconazole (MIC = 16 μg/ml), and amphotericin B (MIC = 4 μg/ml) but still susceptible to posaconazole (MIC = 0.008 μg/ml) and caspofungin (MIC = 0.5 μg/ml). In conclusion, MALDI-TOF MS permitted the early selection of an unusual isolate, which was still unreported in molecular databases but could not be unambiguously classified based on phylogenetic approaches.

Isothermal microcalorimetry: a novel method for real-time determination of antifungal susceptibility of Aspergillus species.

We evaluated microcalorimetry for real-time susceptibility testing of Aspergillus spp. based on growth-related heat production. The minimal heat inhibitory concentration (MHIC) for A. fumigatus ATCC 204305 was 1 mg/L for amphotericin B, 0.25 mg/L for voriconazole, 0.06 mg/L for posaconazole, 0.125 mg/L for caspofungin and 0.03 mg/L for anidulafungin. Agreement within two 2-fold dilutions between MHIC (determined by microcalorimetry) and MIC or MEC (determined by CLSI M38A) was 90% for amphotericin B, 100% for voriconazole, 90% for posaconazole and 70% for caspofungin. This proof-of-concept study demonstrated the potential of isothermal microcalorimetry for growth evaluation of Aspergillus spp. and real-time antifungal susceptibility testing.