RESUMEN
Previous studies have correlated the severity of recurrent vulvovaginal Candida infections (VVC) and balanitis in patients from China with the presence of some dominant genotypes at the ORF RLM1. Here we tested VVC vs non-VVC isolates from Portugal, Brazil and Greece and, although the same genotypes were identified in VVC isolates, they were present in only five out of 150 strains. However, this analysis showed that VVC isolates presented a higher percentage of genotypes with similar high molecular weight alleles, in comparison with strains isolated from other biological sources.
Asunto(s)
Alelos , Candida albicans/genética , Candidiasis Vulvovaginal/microbiología , Variación Genética , Factores de Transcripción/genética , Brasil , Candida albicans/clasificación , Candida albicans/aislamiento & purificación , Candidiasis/microbiología , ADN de Hongos/genética , Femenino , Proteínas Fúngicas/genética , Frecuencia de los Genes , Estudios de Asociación Genética , Genotipo , Grecia , Humanos , Repeticiones de Microsatélite , Peso Molecular , PortugalRESUMEN
In this study, PCR fingerprinting using the universal primer T3B was applied to distinguish among clinical and environmental species of the Sporothrix complex, Sporothrix brasiliensis, S. globosa, S. mexicana, S. pallida, S. luriei and S. schenckii sensu stricto. The T3B fingerprinting generated clearly distinct banding patterns, allowing the correct identification of all 43 clinical and environmental isolates at the species level, what was confirmed by partial calmodulin gene sequence analyses. This technique is reproducible and provides the identification of all species of the Sporothrix complex with sufficient accuracy to be applied in clinical mycology laboratories as well as in epidemiological studies in order to obtain a better understanding of the epidemiology of sporotrichosis.
Asunto(s)
Dermatoglifia del ADN/métodos , Técnicas de Tipificación Micológica , Sporothrix/clasificación , Sporothrix/aislamiento & purificación , Calmodulina , Cartilla de ADN , ADN de Hongos/genética , Filogenia , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN , Sporothrix/genética , Esporotricosis/epidemiologíaRESUMEN
Nosocomial fungal bloodstream infections (BSI) are increasing significantly in hospitalized patients and Candida parapsilosis has emerged as an important pathogen responsible for numerous outbreaks. The objective of this study was to evaluate C. parapsilosis sensu lato infection scenarios, regarding species distribution and strain relatedness. One hundred isolates of C. parapsilosis sensu lato derived from blood cultures and catheter tips were analysed by multiplex microsatellite typing and by sequencing D1/D2 regions of the ribosomal DNA. Our results indicate that 9.5â% of patients presented infections due to C. parapsilosis and Candida orthopsilosis, 57.1â% due to C. parapsilosis, 28.3â% due to C. orthopsilosis and 4.8â% due to Candida metapsilosis. Eighty per cent of the C. parapsilosis BSIs were due to a single strain that was also identified in the catheter, but in 10â% of the cases C. parasilosis was identified in the catheter but the BSI was due to C. orthopsilosis. There is a significant probability that C. parapsilosis isolates collected from the same patient at more than 3 months interval are of different strains (Pâ=â0.0179). Moreover, several isolates were identified persistently in the same hospital, infecting six different patients. The incidence of polyfungal BSI infections with C. parapsilosis and C. orthopsilosis is reported herein for the first time, emphasizing the fact that the species identified in the catheter is not always responsible for the BSI, thus impacting the treatment strategy. The observation that strains can remain in the hospital environment for years highlights the possible existence of reservoirs and reinforces the need for accurate genotyping tools, such as the markers used for elucidating epidemiological associations and detecting outbreaks.
Asunto(s)
Candida/clasificación , Candida/aislamiento & purificación , Candidemia/microbiología , Candidemia/patología , Coinfección/microbiología , Coinfección/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Sangre/microbiología , Candida/genética , Infecciones Relacionadas con Catéteres/microbiología , Infecciones Relacionadas con Catéteres/patología , Catéteres/microbiología , Niño , Preescolar , Análisis por Conglomerados , ADN de Hongos/química , ADN de Hongos/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Femenino , Humanos , Incidencia , Lactante , Recién Nacido , Masculino , Repeticiones de Microsatélite , Persona de Mediana Edad , Epidemiología Molecular , Datos de Secuencia Molecular , Tipificación Molecular , Técnicas de Tipificación Micológica , Filogenia , ARN Ribosómico 28S/genética , Análisis de Secuencia de ADN , Adulto JovenRESUMEN
Microbial culture collections underpin biotechnology applications and are important resources for clinical microbiology by supplying reference strains and/or performing microbial identifications as a service. Proteomic profiles by MALDI-TOF MS have been used for Candida spp. identification in clinical laboratories and demonstrated to be a fast and reliable technique for the routine identification of pathogenic yeasts. The main aim of this study was to apply MALDI-TOF MS combined with classical phenotypic and molecular approaches to identify Candida clinical isolates preserved from 1 up to 52 years in a Brazilian culture collection and assess its value for the identification of yeasts preserved in this type of collections. Forty Candida spp. clinical isolates were identified by morphological and biochemical analyses. Identifications were also performed by the new proteomic approach based on MALDI-TOF MS. Results demonstrated 15% discordance when compared with morphological and biochemical analyses. Discordant isolates were analysed by ITS sequencing, which confirmed the MALDI-TOF MS identifications and these strains were renamed in the culture collection catalogue. In conclusion, proteomic profiles by MALDI-TOF MS represents a rapid and reliable method for identifying clinical Candida species preserved in culture collections and may present clear benefits when compared with the performance of existing daily routine methods applied at health centres and hospitals.
Asunto(s)
Candida/química , Candida/clasificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Brasil , Candida/genética , Candida/aislamiento & purificación , Candidiasis/microbiología , Análisis por Conglomerados , ADN de Hongos/química , ADN de Hongos/genética , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , Humanos , Datos de Secuencia Molecular , Técnicas de Tipificación Micológica , Filogenia , Análisis de Secuencia de ADNRESUMEN
Microbial culture collections underpin biotechnology applications and are important resources for clinical microbiology by supplying reference strains and/or performing microbial identifications as a service. Proteomic profiles by MALDI-TOF MS have been used for Candida spp. identification in clinical laboratories and demonstrated to be a fast and reliable technique for the routine identification of pathogenic yeasts. The main aim of this study was to apply MALDI-TOF MS combined with classical phenotypic and molecular approaches to identify Candida clinical isolates preserved from 1 up to 52 years in a Brazilian culture collection and assess its value for the identification of yeasts preserved in this type of collections. Forty Candida spp. clinical isolates were identified by morphological and biochemical analyses. Identifications were also performed by the new proteomic approach based on MALDI-TOF MS. Results demonstrated 15% discordance when compared with morphological and biochemical analyses. Discordant isolates were analysed by ITS sequencing, which confirmed the MALDI-TOF MS identifications and these strains were renamed in the culture collection catalogue. In conclusion, proteomic profiles by MALDI-TOF MS represents a rapid and reliable method for identifying clinical Candida species preserved in culture collections and may present clear benefits when compared with the performance of existing daily routine methods applied at health centres and hospitals.
Asunto(s)
Humanos , Candida/química , Candida/clasificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Brasil , Análisis por Conglomerados , Candida/genética , Candida/aislamiento & purificación , Candidiasis/microbiología , ADN de Hongos/genética , ADN Espaciador Ribosómico/genética , Datos de Secuencia Molecular , Técnicas de Tipificación Micológica , FilogeniaRESUMEN
Microbial culture collections underpin biotechnology applications and are important resources for clinical microbiology by supplying reference strains and/or performing microbial identifications as a service. Proteomic profiles by MALDI-TOF MS have been used for Candida spp. identification in clinical laboratories and demonstrated to be a fast and reliable technique for the routine identification of pathogenic yeasts. The main aim of this study was to apply MALDI-TOF MS combined with classical phenotypic and molecular approaches to identify Candida clinical isolates preserved from 1 up to 52 years in a Brazilian culture collection and assess its value for the identification of yeasts preserved in this type of collections. Forty Candida spp. clinical isolates were identified by morphological and biochemical analyses. Identifications were also performed by the new proteomic approach based on MALDI-TOF MS. Results demonstrated 15% discordance when compared with morphological and biochemical analyses. Discordant isolates were analysed by ITS sequencing, which confirmed the MALDI-TOF MS identifications and these strains were renamed in the culture collection catalogue. In conclusion, proteomic profiles by MALDI-TOF MS represents a rapid and reliable method for identifying clinical Candida species preserved in culture collections and may present clear benefits when compared with the performance of existing daily routine methods applied at health centres and hospitals.
Asunto(s)
Humanos , Candida/química , Candida/clasificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Brasil , Análisis por Conglomerados , Candida/genética , Candida/aislamiento & purificación , Candidiasis/microbiología , ADN de Hongos/química , ADN de Hongos/genética , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , Datos de Secuencia Molecular , Técnicas de Tipificación Micológica , Filogenia , Análisis de Secuencia de ADNRESUMEN
This article describes PCR fingerprinting using the universal primer T3B to distinguish among species of the Sporothrix complex, S. brasiliensis, S. globosa, S. mexicana, and S. schenckii. This methodology generated distinct banding patterns, allowing the correct identification of all 35 clinical isolates at the species level, confirmed by partial calmodulin (CAL) gene sequence analyses. This methodology is simple, reliable, rapid, and cheap, making it an ideal routine identification system for clinical mycology laboratories.
Asunto(s)
Dermatoglifia del ADN/métodos , Tipificación Molecular/métodos , Reacción en Cadena de la Polimerasa/métodos , Sporothrix/clasificación , Sporothrix/aislamiento & purificación , Calmodulina/genética , Cartilla de ADN/genética , ADN de Hongos/química , ADN de Hongos/genética , Humanos , Datos de Secuencia Molecular , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Sporothrix/genéticaRESUMEN
Candida albicans population studies showed that this species could be divided into sub-groups of closely related strains, designated by clades. Since the emergence of microsatellite analysis as a PCR based method, this technique has been successfully used as a tool to differentiate C. albicans isolates but has never been tested regarding clustering of the five major clades. In this study we tested microsatellite length polymorphism (MLP) ability to group 29 C. albicans strains previously defined as belonging to clades I, II, III, E and SA, nine atypical strains from Angola and Madagascar, and 78 Portuguese clinical isolates. MLP typing of the total 116 strains analyzed yielded 87 different multilocus allelic combinations which resulted in a high discriminatory power index, of 0.987, with only two markers, CA1 and CEF3. Cluster analysis of the 29 previously defined strains grouped them according to their clade designation with a matrix cophenetic correlation of r=0.963 after a normalized Mantel statistic. Clustering analysis of the 116 strains maintained the same groupings, clearly defining the five major C. albicans clades. The cophenetic value obtained was of r=0.839, and the one-tail probability of the normalized Mantel statistic out of 1000 random permutations was P=0.0020. The proportion of Portuguese isolates in the groups I, II, III and SA was of 2.7%, 15.4%, 3.8% and 0%, respectively. None of the isolates co-clustered with the atypical strains. These results confirm MLP typing as a good method both to type and differentiate C. albicans isolates and to group isolates, identifying the major C. albicans clades, similarly to Ca3 fingerprinting and multilocus sequence typing (MLST).