[Antifungal susceptibility testing in yeasts. Update and novelties]. / Estudios de sensibilidad en levaduras. Actualización y novedades.

This text reviews and updates the uses of the reference procedures for antifungal susceptibility testing in yeasts, the reliability of the commercial methods and the guidelines for the use of these procedures for patient management and for epidemiological reasons to determine the susceptibility profile and the emergence of resistances. Novelties in the procedures of setting clinical breakpoints of antifungal agents by both the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the US Clinical Laboratory Standards Institute (CLSI) are also reviewed.

In vitro activity of nine antifungal agents against clinical isolates of Aspergillus calidoustus.

This study analyzes Aspergillus isolates from the Spanish National Centre for Microbiology collection, which were identified morphologically as members of Aspergillus section Usti. Strains were identified through the analysis of the Internal Transcribed Spacer regions and partial beta tubulin gene sequences. One Aspergillus pseudodeflectus isolate and eight Aspergillus calidoustus isolates were detected in this panel of clinical strains. Terbinafine and the echinocandins micafungin and anidulafungin, were the drugs most active against these species.

Antifungal susceptibility profile in vitro of Sporothrix schenckii in two growth phases and by two methods: microdilution and E-test.

The susceptibility profile of 91 Sporothrix schenckii isolates in both growth phases was determined by microdilution test (Antifungal Susceptibility Testing of the European Committee for Antimicrobial Susceptibility Testing; AFST-EUCAST). Amphotericin B (AMB), itraconazole (ITC), posaconazole, ravuconazole and terbinafine were found active in vitro against both phases but minimum inhibitory concentrations values for mycelial phase were significantly higher. Fluconazole (FLC) and voriconazole (VRC) were inactive in vitro against both phases. The E-test technique was also performed with 41 representative isolates for AMB, FLC, ITC and VRC. Average agreement rates between yeast phase microdilution results and E-test results were high for AMB (77.5%) and FLC (87.8%), but low for ITC and VRC with rates of 56.4% and 54.5%, respectively. AFST-EUCAST is not the most recommended test to perform drug susceptibility testing of S. schenckii in clinical laboratories, and E-test could be an alternative methodology for this purpose, mainly when the activity in vitro of antifungal agents of AMB and FLC are evaluated.

Activity of posaconazole and other antifungal agents against Mucorales strains identified by sequencing of internal transcribed spacers.

The antifungal susceptibility profiles of 77 clinical strains of Mucorales species, identified by internal transcribed spacer sequencing, were analyzed. MICs obtained at 24 and 48 h were compared. Amphotericin B was the most active agent against all isolates, except for Cunninghamella and Apophysomyces isolates. Posaconazole also showed good activity for all species but Cunninghamella bertholletiae. Voriconazole had no activity against any of the fungi tested. Terbinafine showed good activity, except for Rhizopus oryzae, Mucor circinelloides, and Rhizomucor variabilis isolates.

Activity profile in vitro of micafungin against Spanish clinical isolates of common and emerging species of yeasts and molds.

A collection of 2,278 isolates belonging to 86 different fungal species was tested with micafungin and eight other drugs using the EUCAST procedures. Micafungin was active against species of Candida and Aspergillus (even azole-resistant species) as well as Penicillium spp., Scedosporium apiospermum, and Acremonium spp. It was inactive for species of Basidiomycota and Mucorales and for multiresistant species such as those of Fusarium.

Susceptibility testing and molecular classification of Paecilomyces spp.

In vitro susceptibility profiles of 58 Paecilomyces clinical isolates are reported. Amphotericin B, itraconazole, and echinocandins showed poor activity against Paecilomyces lilacinus, while the new triazoles were active against it. Paecilomyces variotii exhibited a different susceptibility pattern, being susceptible to most antifungal agents apart from voriconazole and ravuconazole.

Epidemiological cutoffs and cross-resistance to azole drugs in Aspergillus fumigatus.

Antifungal susceptibility testing of molds has been standardized in Europe and in the United States. Aspergillus fumigatus strains with resistance to azole drugs have recently been detected and the underlying molecular mechanisms of resistance characterized. Three hundred and ninety-three isolates, including 32 itraconazole-resistant strains, were used to define wild-type populations, epidemiological cutoffs, and cross-resistance between azole drugs. The epidemiological cutoff for itraconazole, voriconazole, and ravuconazole for the wild-type populations of A. fumigatus was < or =1 mg/liter. For posaconazole, the epidemiological cutoff was < or =0.25 mg/liter. Up till now, isolates susceptible to itraconazole have not yet displayed resistance to other azole drugs. Cross-resistance between azole drugs depends on specific mutations in cyp51A. Thus, a substitution of glycine in position 54 of Cyp51A confers cross-resistance between itraconazole and posaconazole. A substitution of methionine at position 220 or a duplication in tandem of a 34-bp fragment in the cyp51A promoter combined with a substitution of leucine at position 98 for histidine confers cross-resistance to all azole drugs tested. The results obtained in this study will help to develop clinical breakpoints for azole drugs and A. fumigatus.

Antifungal susceptibility profile of clinical Fusarium spp. isolates identified by molecular methods.

OBJECTIVES: To analyse the susceptibility pattern of a collection of Fusarium clinical isolates. METHODS: The antifungal susceptibility pattern of 67 isolates of Fusarium was analysed. Strains were identified by morphological and molecular methods by means of sequencing elongation factor alpha. RESULTS AND CONCLUSIONS: Six different species were identified. Fusarium solani was the most frequently isolated, followed by Fusarium oxysporum, Fusarium proliferatum and Fusarium verticilloides. Amphotericin B was the only drug with in vitro activity (range: 0.015-32 mg/L). The rest of the antifungals tested (itraconazole, voriconazole, ravuconazole, posaconazole and terbinafine) showed very poor activity against Fusarium, confirming the multiresistant nature of this genus.

Clinical relevance of resistance to antifungals.

The standardization of antifungal sensitivity tests represents a huge advance in the detection of antifungal drug resistance. Thus, the reference methods of the European Committee on Antibiotic Susceptibility Testing and the Clinical Laboratory Standards Institute have proven capable of detecting strains of yeasts and filamentous fungi with high minimum inhibitory concentrations (MIC) to antifungal agents. This standardization has enabled the genetic alterations responsible for the high MICs to be studied at the molecular level. Furthermore, these strains have been used in experimental models to obtain pharmacokinetic parameters that may allow us to predict clinical response. However, the correlation of the course of the infection in humans with the sensitivity or resistance of the strain is a controversial area with many unanswered questions. We analyze whether the MICs of human pathogenic fungi have clinical relevance, that is, if they affect the course of the infection.

Scedosporium apiospermum and S. prolificans mixed disseminated infection in a lung transplant recipient: An unusual case of long-term survival with combined systemic and local antifungal therapy in intensive care unit.

Infections due Scedosporium spp. in lung transplant recipients are associated with disseminated disease with high mortality rates. The adjunctive local antifungal therapy may be a useful option when systemic treatment is insufficient and/or surgery is not feasible. We present a case of mixed disseminated infection due Scedosporium apiospermum and S. prolificans in a lung transplant recipient. Combined local and systemic antifungal therapy provided an unusual long-term survival in the intensive care unit.

Fungal necrotizing fasciitis, an emerging infectious disease caused by Apophysomyces (Mucorales).

BACKGROUND: The mucoralean fungi are emerging causative agents of primary cutaneous infections presenting in the form of necrotizing fasciitis. AIMS: The aim of this study was to investigate a series of suspected necrotizing fasciitis cases by Apophysomyces species over one-year period in a northern Indian hospital. METHODS: The clinical details of those patients suspected to suffer from fungal necrotizing fasciitis were recorded. Skin biopsies from local wounds were microscopically examined and fungal culturing was carried out on standard media. The histopathology was evaluated using conventional methods and special stains. Apophysomyces isolates were identified by their morphology and by molecular sequencing of the internal transcribed spacer (ITS) region of the ribosomal genes. Antifungal susceptibility testing was carried out following EUCAST guidelines and treatment progress was monitored. RESULTS: Seven patients were found to be suffering from necrotizing fasciitis caused by Apophysomyces spp. Six isolates were identified as Apophysomyces variabilis and one as Apophysomyces elegans. Five patients had previously received intramuscular injections in the affected area. Three patients recovered, two died and the other two left treatment against medical advice and are presumed to have died due to their terminal illnesses. Posaconazole and terbinafine were found to be the most active compounds against A. variabilis, while the isolate of A. elegans was resistant to all antifungals tested. CONCLUSIONS: Apophysomyces is confirmed as an aggressive fungus able to cause fatal infections. All clinicians, microbiologists and pathologists need to be aware of these emerging mycoses as well as of the risks involved in medical practices, which may provoke serious fungal infections such as those produced by Apophysomyces.

Fungal necrotizing fasciitis, an emerging infectious disease caused by Apophysomyces (Mucorales) / Fascitis necrotizante fúngica, una enfermedad infecciosa emergente causada por Aphophysomyces (Mucorales)

Background: The mucoralean fungi are emerging causative agents of primary cutaneous infections presenting in the form of necrotizing fasciitis. Aims: The aim of this study was to investigate a series of suspected necrotizing fasciitis cases by Apophysomycesspecies over one-year period in a northern Indian hospital. Methods: The clinical details of those patients suspected to suffer from fungal necrotizing fasciitis were recorded. Skin biopsies from local wounds were microscopically examined and fungal culturing was carried out on standard media. The histopathology was evaluated using conventional methods and special stains.Apophysomyces isolates were identified by their morphology and by molecular sequencing of the internal transcribed spacer (ITS) region of the ribosomal genes. Antifungal susceptibility testing was carried out following EUCAST guidelines and treatment progress was monitored. Results: Seven patients were found to be suffering from necrotizing fasciitis caused by Apophysomyces spp. Six isolates were identified as Apophysomyces variabilis and one as Apophysomyces elegans. Five patients had previously received intramuscular injections in the affected area. Three patients recovered, two died and the other two left treatment against medical advice and are presumed to have died due to their terminal illnesses. Posaconazole and terbinafine were found to be the most active compounds against A. variabilis, while the isolate of A. elegans was resistant to all antifungals tested. Conclusions: Apophysomyces is confirmed as an aggressive fungus able to cause fatal infections. All clinicians, microbiologists and pathologists need to be aware of these emerging mycoses as well as of the risks involved in medical practices, which may provoke serious fungal infections such as those produced byApophysomyces (AU)

Antecedentes: Los hongos mucorales son agentes emergentes causantes de infecciones cutáneas primarias presentes en forma de fascitis necrotizante. Objetivos: La finalidad de este estudio fue la de investigar una serie de infecciones sugestivas de fascitis necrotizante causadas por alguna de las especies de Apophysomyces a lo largo de un año en un hospital del norte de la India. Métodos: Se obtuvieron los datos de todos los pacientes con sospecha de fascistis necrotizante. Las biopsias de piel de la zona afectada fueron cultivadas en medios de cultivos estándar y se evaluaron histopatológicamente mediante tinciones convencionales y específicas para hongos. Los aislamientos de Apophysomyces fueron identificados morfológicamente y mediante la secuenciación del espaciador intergénico ribosomal (ITS). La sensibilidad antifúngica se determinó mediante el método EUCAST y la evolución de los pacientes fue monitorizada. Resultados: Se encontraron siete pacientes con fascitis necrotizante causada por especies de Apophysomyces. Seis aislamientos fueron identificados como Apophysomyces variabilis y uno como Apophysomyces elegans. Cinco pacientes habían recibido previamente inyecciones intramusculares en el área afectada. Tres pacientes se recuperaron, dos fallecieron y de los dos restantes no se tiene seguimiento médico, aunque presumiblemente fallecieron debido a que padecían enfermedades terminales. El posaconazol y la terbinafina fueron los compuestos más activos frente a A. variabilis, mientras que el único aislamiento deA. elegans fue resistente a todos los antifúngicos ensayados. Conclusiones: Se confirma que Apophysomyces es un hongo agresivo capaz de causar infecciones con desenlace fatal. Clínicos, microbiólogos y patólogos deben ser conscientes de los riesgos de estas micosis emergentes y de que determinadas prácticas médicas puedan provocar infecciones fúngicas graves como las producidas por Apophysomyces (AU)

Molecular epidemiology and antifungal susceptibility patterns of Sporothrix schenckii isolates from a cat-transmitted epidemic of sporotrichosis in Rio de Janeiro, Brazil.

Since 1998 a cat-transmitted epidemic of sporotrichosis has been observed in Rio de Janeiro (RJ), Brazil. Besides the lymphocutaneous and fixed forms, other presentations, such as disseminated cutaneous and mucosal involvement, as well as for the first time, erythema nodosum and erythema multiforme have been reported associated with sporotrichosis. This study investigates the phenotypes and genotypes of Sporothrix schenckii isolates recovered from different clinical forms of the disease noted as part of this epidemic. A total of 88 isolates recovered from 59 cases associated with the epidemic and 29 controls (from cases in other Brazilian regions and Spain) were included in this study. In vitro susceptibility testing was conducted as part of the phenotypic analysis, while the genotypic analysis involved a DNA fingerprinting method with primer M13 and ribosomal DNA sequencing of the internal transcribed spacer (ITS). MIC values of amphotericin B, itraconazole, posaconazole, ravuconazole, and terbinafine were found to be significantly lower (P<0.01) for isolates associated with the epidemic than for control strains. No differences in MIC values were observed related to clinical forms of the infection. Fingerprinting analysis showed that RJ epidemic strains were genetically related. Although nine subtypes were found, they were not associated with specific clinical forms. Similar results were obtained with the ITS sequence analysis. These data suggest that the strains isolated from the epidemic cases of sporotrichosis in RJ all originated from a common source.

Time of incubation for antifungal susceptibility testing of Aspergillus fumigatus: can MIC values be obtained at 24 hours?

A collection of Aspergillus fumigatus isolates was used to check if MICs can be read at 24 h. At 24 h, the geometric mean MIC of itraconazole for resistant isolates was determined to be 5.11 mg/liter, but the MIC was read as 16 mg/liter at 48 h. At 24 h, MICs for 51.5% of resistant strains were determined to be

[Assessment of a quantitative PCR method for clinical diagnosis of imported histoplasmosis]. / Evaluación de una técnica de PCR cuantitativa para el diagnóstico clínico de la histoplasmosis importada.

OBJECTIVE: Evaluation of the usefulness of a quantitative real-time polymerase chain reaction-based (RT-PCR) technique for clinical diagnosis of histoplasmosis. METHODS: Primers and probes were designed on the basis of sequences from the ITS regions of ribosomal DNA of 20 clinical strains of Histoplasma capsulatum. LightCycler procedures (Roche Applied Science) were used with probes marked by fluorescence resonance energy transfer (FRET). Reproducibility, sensitivity, and specificity were analyzed. In addition, an internal control was designed to identify false negative results by PCR inhibition. The RT-PCR assay was tested in 22 clinical samples from 14 patients with proven histoplasmosis. In addition, 30 samples from patients with febrile neutropenia or mycoses other than histoplasmosis, and from healthy volunteers were analyzed as controls. RESULTS: The limit of detection of the assay was 1 fg of genomic DNA per microl of sample. The PCR-based technique was reproducible and highly specific. Positive results were obtained in 11/14 (78.6%) patients and in 17/22 (77.3%) clinical samples. RT-PCR was positive in 100% of respiratory secretions and bone marrow samples, but only 70% of sera (p < 0.01). Mean fungal DNA value was 23.1 fg/microl in serum and 4.85 x 10(3) fg/microl in respiratory and bone marrow samples. RT-PCR results were positive in serum from three HIV patients for which antibody detection by immunodiffusion was negative. Specificity was 100%, since PCR results were negative for all the control samples. CONCLUSION: Thes RT-PCR technique is a sensitive, specific method for early diagnosis of histoplasmosis, particularly when respiratory secretions or bone marrow samples are analyzed. The reliability is lower in serum, but it can be used as an additional, complementary technique to culture and serology in HIV patients.

Prevalence and susceptibility testing of new species of pseudallescheria and scedosporium in a collection of clinical mold isolates.

The prevalence of new species of Pseudallescheria and Scedosporium in a collection of 46 clinical isolates was analyzed. Strain identification was done by morphological and molecular methods. Four Scedosporium aurantiacum isolates were detected among the panel of clinical strains. The susceptibility profile of S. aurantiacum was similar to that of Scedosporium apiospermum.

Head-to-head comparison of the activities of currently available antifungal agents against 3,378 Spanish clinical isolates of yeasts and filamentous fungi.

We have compared the activities of posaconazole and other currently available antifungal agents against a collection of 3,378 clinical isolates of yeasts and filamentous fungi. A total of 1,997 clinical isolates of Candida spp., 359 of other yeast species, 697 strains of Aspergillus spp., and 325 nondermatophyte non-Aspergillus spp. were included. The average geometric means of the MICs of agents that were tested against Candida spp. were 0.23 microg/ml for amphotericin B, 0.29 microg/ml for flucytosine, 0.97 microg/ml for fluconazole, 0.07 microg/ml for itraconazole, 0.04 microg/ml for voriconazole, 0.15 microg/ml for caspofungin, and 0.03 microg/ml for posaconazole. Voriconazole and posaconazole were active in vitro against the majority of isolates, with resistance to fluconazole and itraconazole, and against Cryptococcus neoformans and other Basidiomycota yeasts. Posaconazole was the most active of antifungal agents tested against Aspergillus spp., with an average geometric mean of 0.10 microg/ml. It was active against Paecilomyces spp., Penicillium spp., Scedosporium apiospermum, and some black fungi, such as Alternaria spp. Multiresistant filamentous fungi, such as Scedosporium prolificans, Scopulariopsis brevicaulis, and Fusarium solani, were also resistant to voriconazole, caspofungin, and posaconazole. Amphotericin B and posaconazole were found to be active against most of the Mucorales strains tested. Posaconazole and currently available antifungal agents exhibit a potent activity in vitro against the majority of pathogenic fungal species.

In vitro activity of ravuconazole against 923 clinical isolates of nondermatophyte filamentous fungi.

The in vitro activities of ravuconazole against 575 clinical strains of Aspergillus spp. and 348 nondermatophyte non-Aspergillus spp. were analyzed. Ravuconazole was active against Aspergillus spp., other hyaline filamentous fungi, black molds, and some Mucorales. Species such as Scedosporium prolificans, Fusarium spp., and Scopulariopsis spp. were resistant to the triazole.

Scopulariopsis brevicaulis, a fungal pathogen resistant to broad-spectrum antifungal agents.

The antifungal susceptibility results for 32 clinical isolates of Scopulariopsis brevicaulis are presented. Flucytosine and itraconazole were inactive in vitro, and MICs of amphotericin B, voriconazole, and terbinafine for all isolates were high, with geometric means of 13, 25.8, and 14.4 microg/ml, respectively.