RESUMEN
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.
RESUMEN
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)
Asunto(s)
Humanos , Mucorales/patogenicidad , Mucormicosis/epidemiología , Fascitis Necrotizante/epidemiología , /epidemiología , Antifúngicos/uso terapéutico , Estudios ProspectivosRESUMEN
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.
Asunto(s)
Enfermedades Transmisibles Emergentes/microbiología , Dermatomicosis/microbiología , Fascitis Necrotizante/microbiología , Mucorales/aislamiento & purificación , Mucormicosis/microbiología , Infecciones Oportunistas/microbiología , Adulto , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Enfermedades Transmisibles Emergentes/epidemiología , Infección Hospitalaria/tratamiento farmacológico , Infección Hospitalaria/epidemiología , Infección Hospitalaria/microbiología , Infección Hospitalaria/cirugía , ADN de Hongos/genética , ADN Espaciador Ribosómico/genética , Desbridamiento , Dermatomicosis/tratamiento farmacológico , Dermatomicosis/epidemiología , Dermatomicosis/cirugía , Complicaciones de la Diabetes/microbiología , Fascitis Necrotizante/epidemiología , Femenino , Humanos , India/epidemiología , Inyecciones Intramusculares/efectos adversos , Masculino , Persona de Mediana Edad , Mucorales/efectos de los fármacos , Mucorales/genética , Mucorales/patogenicidad , Mucormicosis/tratamiento farmacológico , Mucormicosis/epidemiología , Mucormicosis/cirugía , Técnicas de Tipificación Micológica , Infecciones Oportunistas/tratamiento farmacológico , Infecciones Oportunistas/epidemiología , Infecciones Oportunistas/cirugía , Estudios Prospectivos , Ribotipificación , Análisis de Supervivencia , Infección de Heridas/tratamiento farmacológico , Infección de Heridas/epidemiología , Infección de Heridas/microbiología , Infección de Heridas/cirugíaRESUMEN
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.
Asunto(s)
Levaduras/efectos de los fármacos , Humanos , Pruebas de Sensibilidad MicrobianaRESUMEN
En esta revisión se incluye una actualización sobre las aplicaciones de las técnicas de referencia de los estudios de sensibilidad en levaduras, sobre la utilidad de las técnicas comerciales, y sobre las recomendaciones e indicaciones para realizar estos estudios en la práctica clínica y en programas de vigilancia epidemiológica para conocer la aparición de resistencias. Asimismo se revisan las últimas novedades en el proceso de definición de los puntos de corte, para interpretar los estudios de sensibilidad a los antifúngicos que están llevando a cabo tanto el European Committee on Antimicrobial Susceptibility Testing (EUCAST) como el Clinical Laboratory Standards Institute estadounidense (CLSI)
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
Asunto(s)
Humanos , Levaduras , Técnicas Microbiológicas , Pruebas de Sensibilidad MicrobianaAsunto(s)
Cunninghamella/clasificación , Cunninghamella/aislamiento & purificación , Huésped Inmunocomprometido , Mucormicosis/diagnóstico , Mucormicosis/microbiología , ADN de Hongos/química , ADN de Hongos/genética , ADN Ribosómico/química , ADN Ribosómico/genética , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , Humanos , Masculino , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Adulto JovenRESUMEN
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.
Asunto(s)
Antifúngicos/farmacología , Aspergilosis/microbiología , Aspergillus/efectos de los fármacos , Aspergillus/aislamiento & purificación , Aspergillus/clasificación , ADN de Hongos/química , ADN de Hongos/genética , ADN Ribosómico/química , ADN Ribosómico/genética , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , Proteínas Fúngicas/genética , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , España , Tubulina (Proteína)/genéticaRESUMEN
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.
Asunto(s)
Antifúngicos/farmacología , Sporothrix/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Micelio/efectos de los fármacos , Sporothrix/aislamiento & purificación , Esporotricosis/microbiología , Levaduras/efectos de los fármacosRESUMEN
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.
Asunto(s)
Antifúngicos/farmacología , Equinocandinas/farmacología , Hongos/efectos de los fármacos , Lipopéptidos/farmacología , Acremonium/efectos de los fármacos , Aspergillus/efectos de los fármacos , Candida/clasificación , Candida/efectos de los fármacos , Farmacorresistencia Fúngica , Hongos/clasificación , Hongos/aislamiento & purificación , Humanos , Micafungina , Pruebas de Sensibilidad Microbiana/métodos , Pruebas de Sensibilidad Microbiana/normas , Micosis/microbiología , Penicillium/efectos de los fármacos , Scedosporium/efectos de los fármacos , EspañaRESUMEN
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.
Asunto(s)
Antifúngicos/farmacología , ADN Espaciador Ribosómico/química , Mucorales/efectos de los fármacos , Triazoles/farmacología , Anfotericina B/farmacología , Pruebas de Sensibilidad Microbiana , Mucorales/genéticaRESUMEN
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.
Asunto(s)
Antifúngicos/uso terapéutico , Farmacorresistencia Fúngica , Hongos/efectos de los fármacos , Micosis/microbiología , Micosis/fisiopatología , Europa (Continente) , Humanos , Pruebas de Sensibilidad Microbiana/normas , Estados UnidosRESUMEN
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.
Asunto(s)
Antifúngicos/farmacología , Paecilomyces/efectos de los fármacos , Paecilomyces/genética , Anfotericina B/farmacología , ADN de Hongos/genética , ADN Ribosómico/genética , Equinocandinas/farmacología , Itraconazol/farmacología , Pruebas de Sensibilidad Microbiana , Paecilomyces/clasificación , FilogeniaRESUMEN
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.
Asunto(s)
Antifúngicos/farmacología , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/genética , Azoles/farmacología , Sustitución de Aminoácidos , Aspergilosis/tratamiento farmacológico , Aspergilosis/epidemiología , Aspergilosis/microbiología , Aspergillus fumigatus/aislamiento & purificación , Sistema Enzimático del Citocromo P-450/genética , Farmacorresistencia Fúngica/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Humanos , Itraconazol/farmacología , Pruebas de Sensibilidad Microbiana/normas , Epidemiología Molecular , Datos de Secuencia Molecular , Pirimidinas/farmacología , Tiazoles/farmacología , Triazoles/farmacología , VoriconazolRESUMEN
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.
Asunto(s)
Sporothrix/genética , Sporothrix/aislamiento & purificación , Esporotricosis/epidemiología , Esporotricosis/transmisión , Animales , Antifúngicos , Brasil , Gatos , ADN Espaciador Ribosómico/análisis , Pruebas de Sensibilidad Microbiana , Epidemiología Molecular , Técnicas de Tipificación Micológica , Esporotricosis/etiología , Esporotricosis/patología , Zoonosis/transmisiónRESUMEN
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.
Asunto(s)
Antifúngicos/farmacología , Fusarium/efectos de los fármacos , Fusarium/aislamiento & purificación , Micosis/microbiología , ADN de Hongos/genética , Farmacorresistencia Fúngica , Fusarium/clasificación , Fusarium/citología , Humanos , Pruebas de Sensibilidad Microbiana , Factor 1 de Elongación Peptídica/genética , Filogenia , Análisis de Secuencia de ADNRESUMEN
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 Asunto(s)
Antifúngicos/farmacología
, Aspergillus fumigatus/efectos de los fármacos
, Pruebas de Sensibilidad Microbiana/métodos
, Reproducibilidad de los Resultados
, Factores de Tiempo
RESUMEN
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.
Asunto(s)
ADN de Hongos/aislamiento & purificación , ADN Ribosómico/aislamiento & purificación , Histoplasma/aislamiento & purificación , Histoplasmosis/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Infecciones Oportunistas Relacionadas con el SIDA/diagnóstico , Infecciones Oportunistas Relacionadas con el SIDA/microbiología , África/etnología , Líquidos Corporales/microbiología , Médula Ósea/microbiología , América Central/etnología , Sistemas de Computación , ADN de Hongos/genética , ADN Ribosómico/genética , Emigración e Inmigración , Infecciones por VIH/complicaciones , Histoplasma/genética , Histoplasmosis/complicaciones , Histoplasmosis/epidemiología , Histoplasmosis/microbiología , Histoplasmosis/transmisión , Inmunodifusión , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , América del Sur/etnología , España/epidemiología , ViajeRESUMEN
Objetivos. Valoración de la utilidad de una técnica de reacción en cadena de la polimerasa (PCR) cuantitativa en tiempo real (PCR-TR) para el diagnóstico de histoplasmosis en muestras clínicas. Métodos. Para el diseño de los iniciadores y de las sondas se analizaron las secuencias de las regiones ITS (internal transcriber spacers) del ADN ribosómico de 20 cepas de Histoplasma capsulatum. Se empleó la tecnología LightCycler (Roche Applied Science) y la sonda fue diseñada mediante el sistema Fluoresce Resonance Energy Transfer (FRET). Se realizaron estudios de reproducibilidad, sensibilidad y especificidad. Además, se incluyó un sistema de control interno para reconocer falsos negativos por inhibición de la reacción de PCR. Tras ello, se testó la técnica en 22 muestras clínicas procedentes de 14 pacientes diagnosticados de histoplasmosis probada. Además se analizaron 30 muestras de control, procedentes de enfermos con otras micosis, de pacientes con neutropenia febril y de voluntarios sanos. Resultados. El límite de detección de la técnica fue de 1 fg de ADN fúngico por ml de muestra. Así mismo fue reproducible y muy específica. La técnica fue positiva en 11 de los 14 enfermos (78,6%) y en 17 de las 22 muestras analizadas (77,3%). Por muestras, el 100% de las muestras respiratorias y de los aspirados de médula ósea fueron positivos, pero sólo el 70% de los sueros (p < 0,01). La cantidad media de ADN detectado en suero fue de 23,1 fg/µl, mientras que en muestras respiratorias y aspirados medulares fue de 4,85 x 10 3 fg/µl (p < 0,01). La técnica de PCR-TR fue positiva en suero de 3 enfermos por el virus de la inmunodeficiencia humana (VIH) positivos, en los que la detección de anticuerpos por inmunodifusión fue negativa. La especificidad fue del 100%, ya que ninguna de las muestras de control dio un resultado positivo. Conclusión. Esta técnica de PCR-TR es un método sensible y específico para el diagnóstico rápido de histoplasmosis, sobre todo en muestras respiratorias y aspirados de médula ósea. La sensibilidad en suero es inferior, pero puede ser complementaria al cultivo y a la serología en enfermos VIH positivos (AU)
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 ml 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/µl in serum and 4.85x10 3 fg/µl 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 (AU)
Asunto(s)
Humanos , ADN de Hongos/aislamiento & purificación , ADN Ribosómico/aislamiento & purificación , Histoplasma/aislamiento & purificación , Histoplasmosis/diagnóstico , Histoplasmosis/epidemiología , Reacción en Cadena de la Polimerasa/métodos , Infecciones Oportunistas Relacionadas con el SIDA/diagnóstico , Infecciones Oportunistas Relacionadas con el SIDA/microbiología , África/etnología , ADN de Hongos/genética , ADN Ribosómico/genética , Infecciones por VIH/complicaciones , Histoplasma/genética , ViajeRESUMEN
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.
Asunto(s)
Pseudallescheria , Scedosporium , Antifúngicos/farmacología , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Filogenia , Pseudallescheria/efectos de los fármacos , Pseudallescheria/genética , Pseudallescheria/aislamiento & purificación , Scedosporium/efectos de los fármacos , Scedosporium/genética , Scedosporium/aislamiento & purificaciónRESUMEN
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.