Antifungal susceptibilities of clinical and environmental isolates of Cryptococcus neoformans in goiânia city, Goiás, Brasil

We evaluated the antifungal activities of amphotericin B, fluconazole, itraconazole and voriconazole in 70 Cryptococcus neoformans strains obtained from cerebrospinal fluid from AIDS patients and 40 C. neoformans strains isolated from the environment. Four clinical isolates were identified as C. neoformans var. gattii. The susceptibility test was done using a broth microdilution method according to NCCLS M27-A2. Range minimal inhibitory concentrations (MICs) for C. neoformans clinical isolates were 0.06-1.0 µg/mL for amphotericin B, 0.125-8 µg/mL for fluconazole, 0.03-0.5 µg/mL for itraconazole and 0.03-0.25 µg/mL for voriconazole. C. neoformans environmental isolates showed range MICs 0.015-0.125 µg/mL, 0.25-2.0 µg/mL, 0.007-0.125 µg/mL and 0.03-0.25 µg/mL for amphotericin B, fluconazole, itraconazole and voriconazole respectively. The MICs results obtained from clinical and environmental isolates showed similar pattern of susceptibility and no resistance has been found in our isolates.

Primary aspergilloma and subacute invasive aspergillosis in two AIDS patients

Although uncommon, invasive aspergillosis in the setting of AIDS is important because of its peculiar clinical presentation and high lethality. This report examines two AIDS patients with a history of severe cellular immunosuppression and previous neutropenia, who developed subacute invasive aspergillosis. One female patient developed primary lung aspergilloma, with dissemination to the mediastinum, vertebrae, and spine, which was fatal despite antifungal treatment. The second patient, who had multiple cavitary brain lesions, and eye and lung involvement, recovered following voriconazole and itraconazole, and drugs for increasing neutrophil and CD4+ lymphocyte levels. These cases demonstrate the importance of Aspergillus infections following neutropenia in AIDS patients, and emphasize the need for early and effective antifungal therapy.

Sporothrix schenckii COMPLEX: SUSCEPTIBILITIES TO COMBINED ANTIFUNGAL AGENTS AND CHARACTERIZATION OF ENZYMATIC PROFILES

SUMMARY Sporothrix schenckiiwas reclassified as a complex encompassing six cryptic species, which calls for the reassessment of clinical and epidemiological data of these new species. We evaluated the susceptibility of Sporothrix albicans (n = 1) , S. brasiliensis (n = 6) , S. globosa (n = 1), S. mexicana(n = 1) and S. schenckii(n = 36) to terbinafine (TRB) alone and in combination with itraconazole (ITZ), ketoconazole (KTZ), and voriconazole (VRZ) by a checkerboard microdilution method and determined the enzymatic profile of these species with the API-ZYM kit. Most interactions were additive (27.5%, 32.5% and 5%) or indifferent (70%, 50% and 52.5%) for TRB+KTZ, TRB+ITZ and TRB+VRZ, respectively. Antagonisms were observed in 42.5% of isolates for the TRB+VRZ combination. Based on enzymatic profiling, the Sporothrix schenckii strains were categorized into 14 biotypes. Leucine arylamidase (LA) activity was observed only for S. albicans and S. mexicana. The species S. globosaand S. mexicanawere the only species without β-glucosidase (GS) activity. Our results may contribute to a better understanding of virulence and resistance among species of the genus Sporothrixin further studies.

Invasive candidosis: contrasting the perceptions of infectious disease physicians and intensive care physicians

Introduction We analyze how infectious disease physicians perceive and manage invasive candidosis in Brazil, in comparison to intensive care unit specialists. Methods A 38-question survey was administered to 56 participants. Questions involved clinicians' perceptions of the epidemiology, diagnosis, treatment and prophylaxis of invasive candidosis. P < 0.05 was considered statistically significant. Results The perception that candidemia not caused by Candida albicans occurs in less than 10% of patients is more commonly held by intensive care unit specialists (p=0.018). Infectious disease physicians almost always use antifungal drugs in the treatment of patients with candidemia, and antifungal drugs are not as frequently prescribed by intensive care unit specialists (p=0.006). Infectious disease physicians often do not use voriconazole when a patient's antifungal treatment has failed with fluconazole, which also differs from the behavior of intensive care unit specialists (p=0.019). Many intensive care unit specialists use fluconazole to treat candidemia in neutropenic patients previously exposed to fluconazole, in contrast to infectious disease physicians (p=0.024). Infectious disease physicians prefer echinocandins as a first choice in the treatment of unstable neutropenic patients more frequently than intensive care unit specialists (p=0.013). When candidemia is diagnosed, most infectious disease physicians perform fundoscopy (p=0.015), whereas intensive care unit specialists usually perform echocardiograms on all patients (p=0.054). Conclusions This study reveals a need to better educate physicians in Brazil regarding invasive candidosis. The appropriate management of this disease depends on more drug options being available in our country in addition to global coverage in private and public hospitals, thereby improving health care.

Environmental isolation, biochemical identification, and antifungal drug susceptibility of Cryptococcus species

Introduction The incidence of opportunistic fungal infections has increased in recent years and is considered an important public health problem. Among systemic and opportunistic mycoses, cryptococcosis is distinguished by its clinical importance due to the increased risk of infection in individuals infected by human immunodeficiency virus. Methods To determine the occurrence of pathogenic Cryptococcus in pigeon excrement in the City of Araraquara, samples were collected from nine environments, including state and municipal schools, abandoned buildings, parks, and a hospital. The isolates were identified using classical tests, and susceptibility testing for the antifungal drugs (fluconazole, itraconazole, voriconazole, and amphotericin B) independently was also performed. After collection, the excrement samples were plated on Niger agar and incubated at room temperature. Results A total of 87 bird dropping samples were collected, and 66.6% were positive for the genus Cryptococcus. The following species were identified: Cryptococcus neoformans (17.2%), Cryptococcus gattii (5.2%), Cryptococcus ater (3.5%), Cryptococcus laurentti (1.7%), and Cryptococcus luteolus (1.7%). A total of 70.7% of the isolates were not identified to the species level and are referred to as Cryptococcus spp. throughout the manuscript. Conclusions Although none of the isolates demonstrated resistance to antifungal drugs, the identification of infested areas, the proper control of birds, and the disinfection of these environments are essential for the epidemiological control of cryptococcosis.

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.

Molecular mechanisms of drug resistance in clinical Candida species isolated from tunisian hospitals.

Antifungal resistance of Candida species is a clinical problem in the management of diseases caused by these pathogens. In this study we identified from a collection of 423 clinical samples taken from Tunisian hospitals two clinical Candida species (Candida albicans JEY355 and Candida tropicalis JEY162) with decreased susceptibility to azoles and polyenes. For JEY355, the fluconazole (FLC) MIC was 8 μg/ml. Azole resistance in C. albicans JEY355 was mainly caused by overexpression of a multidrug efflux pump of the major facilitator superfamily, Mdr1. The regulator of Mdr1, MRR1, contained a yet-unknown gain-of-function mutation (V877F) causing MDR1 overexpression. The C. tropicalis JEY162 isolate demonstrated cross-resistance between FLC (MIC > 128 μg/ml), voriconazole (MIC > 16 μg/ml), and amphotericin B (MIC > 32 μg/ml). Sterol analysis using gas chromatography-mass spectrometry revealed that ergosterol was undetectable in JEY162 and that it accumulated 14α-methyl fecosterol, thus indicating a perturbation in the function of at least two main ergosterol biosynthesis proteins (Erg11 and Erg3). Sequence analyses of C. tropicalis ERG11 (CtERG11) and CtERG3 from JEY162 revealed a deletion of 132 nucleotides and a single amino acid substitution (S258F), respectively. These two alleles were demonstrated to be nonfunctional and thus are consistent with previous studies showing that ERG11 mutants can only survive in combination with other ERG3 mutations. CtERG3 and CtERG11 wild-type alleles were replaced by the defective genes in a wild-type C. tropicalis strain, resulting in a drug resistance phenotype identical to that of JEY162. This genetic evidence demonstrated that CtERG3 and CtERG11 mutations participated in drug resistance. During reconstitution of the drug resistance in C. tropicalis, a strain was obtained harboring only defective Cterg11 allele and containing as a major sterol the toxic metabolite 14α-methyl-ergosta-8,24(28)-dien-3α,6β-diol, suggesting that ERG3 was still functional. This strain therefore challenged the current belief that ERG11 mutations cannot be viable unless accompanied by compensatory mutations. In conclusion, this study, in addition to identifying a novel MRR1 mutation in C. albicans, constitutes the first report on a clinical C. tropicalis with defective activity of sterol 14α-demethylase and sterol Δ(5,6)-desaturase leading to azole-polyene cross-resistance.

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.

Estudi d'utilització d'antifúngics sistèmics a l'Hospital Universitari Vall d'Hebron

Objectius: Descriure les principals característiques de la utilització dels antifúngics sistèmics a l’Hospital Universitari Vall d’Hebron (HUVH) i avaluar la seva adequació. Metodologia: Estudi de prescripció-indicació en pacients majors de 16 anys amb seguiment prospectiu de 40 casos incidents consecutius. Limitats a 10 els casos de fluconazole, es realitza una anàlisi descriptiva ponderada. S’avalua l’adequació segons les recomanacions de la Infectious Diseases Society of America (IDSA). Resultats: El fluconazole va ser el fàrmac més emprat (74,8%; IC del 95%: 60,7-88,9), seguit del voriconazole. L’ús més habitual va ser l’empíric-anticipat (53,4%; IC del 95%: 28,6-78,1) i la indicació més freqüent la profilaxi d’infecció fúngica invasiva (25,0%; IC del 95%: 2,4-47,5). Es van considerar adequades el 71,7% (IC del 95%: 49,1-94,3) de les indicacions, el 100% (IC del 95%: 100-100) dels fàrmacs seleccionats, el 51,2% (IC del 95%: 21,8-80,6) de les dosis i el 61,7% (IC del 95%: 32,6-90,7) de les durades. Conclusions: Els resultats suggereixen que a l’HUVH es fa una prescripció raonada dels antifúngics sistèmics però caldria millorar el seu ús empíricanticipat i específic. Cal interpretar-los amb cautela per la manca de precisió i la dificultat per valorar la situació clínica

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.

Cotrimoxazole enhances the in vitro susceptibility of Coccidioides posadasii to antifungals

The aim of the present study was to evaluate the effect of cotrimoxazole on the in vitro susceptibility of Coccidioides posadasii strains to antifungals. A total of 18 strains of C. posadasii isolated in Brazil were evaluated in this study. The assays were performed in accordance with the Clinical and Laboratory Standards Institute guidelines and the combinations were tested using the checkerboard method. The minimum inhibitory concentrations were reduced by 11, 2.4, 4.3 and 3.5 times for amphotericin B, itraconazole, fluconazole and voriconazole, respectively. Moreover, it was seen that cotrimoxazole itself inhibited C. posadasii strains in vitro. The impairment of folic acid synthesis may be a potential antifungal target for C. posadasii.

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.

Novel point mutations in the ERG11 gene in clinical isolates of azole resistant Candida species

The azoles are the class of medications most commonly used to fight infections caused by Candida sp. Typically, resistance can be attributed to mutations in ERG11 gene (CYP51) which encodes the cytochrome P450 14α-demethylase, the primary target for the activity of azoles. The objective of this study was to identify mutations in the coding region of theERG11 gene in clinical isolates of Candidaspecies known to be resistant to azoles. We identified three new synonymous mutations in the ERG11 gene in the isolates of Candida glabrata (C108G, C423T and A1581G) and two new nonsynonymous mutations in the isolates of Candida krusei - A497C (Y166S) and G1570A (G524R). The functional consequence of these nonsynonymous mutations was predicted using evolutionary conservation scores. The G524R mutation did not have effect on 14α-demethylase functionality, while the Y166S mutation was found to affect the enzyme. This observation suggests a possible link between the mutation and dose-dependent sensitivity to voriconazole in the clinical isolate of C. krusei. Although the presence of the Y166S in phenotype of reduced azole sensitivity observed in isolate C. kruseidemands investigation, it might contribute to the search of new therapeutic agents against resistant Candida isolates.