Selección y optimización de los métodos basados en PCR para la detección de Histoplasma capsulatum var. capsulatum / Selection and optimization of PCR-based methods for the detection of Histoplasma capsulatum var. capsulatum

Antecedentes. Los métodos actuales para el diagnóstico de laboratorio de la histoplasmosis son problemáticos si consideramos los valores de sensibilidad, especificidad y tiempo de ejecución. Objetivos. En este estudio hemos tratado de seleccionar y optimizar los métodos para la detección de Histoplasma capsulatum var. capsulatum mediante la reacción en cadena de la polimerasa (PCR). Métodos. Se evaluaron tres métodos de extracción de ADN y tres métodos de PCR. Se optimizó la concentración de los componentes de esta reacción de PCR y se determinó su co-positividad (sensibilidad) y co-negatividad (especificidad), utilizando muestras de sangre a las que se había añadido H. capsulatum. Resultados. El método de extracción que dio el ADN de más alta calidad utiliza membranas de sílice (DNeasy Blood & Tissue Kit, Qiagen, Hilden, Germany), y el método de PCR con mayor capacidad de detección es el que incluye un gen diana que codifica una proteína de 100 kDa. Nuestra optimización de las condiciones de PCR indicaron que la reacción trabaja en un rango significativo de las concentraciones de componentes y, además, fue capaz de detectar H. capsulatum mejor que las técnicas de cultivo tradicional, con un límite de detección de solo 10 pg de ADN. Conclusiones. En nuestras condiciones experimentales, el método PCR seleccionado en este trabajo (en lugar de PCR anidada) es una herramienta lo suficientemente sensible para el diagnóstico de la histoplasmosis

Background. Current methods for the laboratory diagnosis of histoplasmosis are problematic in terms of their sensitivity, specificity and runtime. Objectives. Thus, in this study, we sought to select and optimize methods for the detection of Histoplasma capsulatum var. capsulatum by polymerase chain reaction (PCR). Methods. Three DNA extraction methods and three PCR methods were evaluated. We optimised the concentration of the components of this PCR reaction and determined its sensitivity and specificity using blood samples to which H. capsulatum had been added. Results. The DNA extraction method that yielded the highest-quality DNA used silica membranes (DNeasy Blood & Tissue Kit, Qiagen, Hilden, Germany), and the amplification method with the best detection capacity used a target gene encoding a 100-kDa protein. Our optimisation of the PCR conditions indicated that the reaction works over a significant range of component concentrations; in addition, it was able to detect H. capsulatum better than traditional culture techniques, with a detection limit of only 10 pg of DNA. Conclusions. In our experimental conditions, the PCR method selected in this work (instead of nested-PCR) is a tool sensitive enough for the diagnosis of histoplasmosis(AU)

Molecular characterisation of the causative agents of Cryptococcosis in patients of a tertiary healthcare facility in the state of Amazonas-Brazil.

As there are four major molecular types of Cryptococcus neoformans (VNI, VNII, VNIII and VNIV) and four molecular types of Cryptococcus gattii (VGI, VGII, VGIII and VGIV), it is important to identify the specific groups causing cryptococcosis in different geographical regions. Here, we investigated the molecular types of 57 cryptococcal isolates from patients in a tertiary care hospital in the state of Amazonas, Brazil, between 2006 and 2010. The isolates were characterised by PCR fingerprinting using the M13 minisatellite and confirmed by URA5-RFLP analysis, and the presence of specific genes from the mating type locus (MATα and MATa) of these species was analysed by PCR. Most of the patients were male (66.7%), between 16 and 30 years of age (51.7%), and HIV-positive (75.0%). Most isolates were collected from cerebrospinal fluid samples (71.7%). Most of the C. neoformans isolates (n=40) were characterised as members of the VNI molecular group (n=39), a unique isolate was characterised as VNII whereas all isolates of C. gattii (n=17) were members of the VGII molecular group. With regard to mating types, 55 isolates were type 'α', and only two were type 'a'. This study revealed the prevalence of the VNI molecular group and provides the first reported observation of the VNII molecular group in the northern region of Brazil.