Polymorphisms and gene expression of metalloproteinases and their inhibitors associated with cerebral ischemic stroke in young patients with sickle cell anemia.

BACKGROUND: Sickle cell anemia (SCA) is a genetic disease with great clinical heterogeneity and few viable strategies for treatment; hydroxyurea (HU) is the only widely used drug. Thus, the study of single nucleotide polymorphisms (SNPs) and the gene expression of MMPs 1, 2, 9, 7 and TIMPs 1 and 2, which are involved in the regulation of extracellular matrix, inflammation, and neuropathies, may provide further insights into the pathophysiology of the disease and elucidate biomarkers and molecules as potential therapeutic targets for patients with SCA. METHODS AND RESULTS: We evaluated 251 young individuals with SCA from northeastern Brazil. The groups were divided according to vaso-occlusive crisis (VOC) and cerebrovascular disease (CVD), compared to control individuals. SNP detection and gene expression assays were performed by real-time PCR, TaqMan system®. Both the expression levels of MMP1 gene, and the SNP MMP1-1607 1G/2G were associated with the risk of cerebral ischemic stroke (IS), and the expression of MMP1 was also associated with a higher frequency of VOC/year. Expression levels of MMP7, TIMP1, and TIMP2 were increased in patients conditioned to IS. The SNP 372T>C (rs4898) TIMP1 T alleles were more frequent in patients with > 5 VOC events/year. The SNP rs17576 of MMP9 showed differences in gene expression levels; it was increased in the genotypes AG, and AG+GG. CONCLUSION: The findings of this study, the SNPs, and expression provide initial support for understanding the role of MMPs-TIMPs in the pathophysiology of SCA in young patients.

Potential role of circulating miR-21 in the diagnosis of hepatocellular carcinoma: a systematic review and meta-analysis.

OBJECTIVE: Identify original articles that analyzed the diagnostic value of miR-21 in hepatocellular carcinoma without language restriction or publication date. METHODOLOGY: We performed structured searches on PubMed, Web of Science, VHL, and EMBASE. The Standard Quality Assessment Criteria for Evaluating Primary Research Papers from a Variety of Fields was used to assess the quality of each study. Random effect models were used to study heterogeneity, evaluated based on the Higgins I2 statistic. RESULTS: 12 articles were evaluated and contained raw data from 1,329 individuals, of which 617 had HCC, 473 were healthy, and 239 had Chronic liver disease. The combined sensitivity and combined specificity of miR-21 for diagnosing HCC were, respectively, 0.83(95% CI:0.78-0.89) and 0.85(95% CI:0.80-0.90). The sensitivity and specificity, in that order, by type of control were 0.81 (95% CI: 0.71-0.91) and 0.88 (95% CI: 0.82-0.93) for CLDs and 0.86(95% CI: 0.81-0.91) and 0.83(95% CI:0.74-0.91) for Healthy controls. CONCLUSION: miR-21 has a moderate overall performance in diagnosing HCC and may serve as a potential non-invasive marker for this early-stage disease. Thus, it may contribute to complementing the results of alpha-fetoprotein in the diagnosis and help to detect HCC at an earlier stage, increasing the survival chances of these patients.

Acolhimento e cuidado à pessoa em uso problemático de drogas / Embracing and caring for problematic drug users / Acogimiento y cuidado a la persona en uso problemático de drogas

Objetivo: analisar práticas de acolhimento na atenção à pessoa em uso problemático de drogas sob o enfoque da integralidade. Método: estudo qualitativo, tendo integralidade como categoria analítica, realizado com 14 usuários e nove profissionais de um Centro de Atenção Psicossocial em 2015, Pernambuco/Brasil. A pesquisa foi aprovada pelo Comitê de Ética em Pesquisa. O material empírico foi produzido por meio de quatro oficinas de reflexão e analisado pela técnica de análise de discurso. Resultados: os discursos de usuários do serviço e de profissionais convergem para práticas de cuidado acolhedoras, capazes de construir relações de confiança e vínculos consistentes, facilitando processos terapêuticos caracterizados pelos dois grupos como resolutivos. Conclusão: O cuidado prestado a pessoas em uso problemático de drogas caminha ao encontro da integralidade, com práticas de acolhimento qualificadas como dialógicas, afetivas e potencial para resolutividade em um contexto favorável à autonomia e ao fortalecimento da cidadania.

Objective: with a comprehensive care focus, to examine practices in care for problematic drug users. Method: this qualitative study of 14 users and nine health professionals at a Psychosocial Care Center in Pernambuco, Brazil, in 2015, was approved by the research ethics committee. The empirical material was produced at four reflection workshops, and analyzed by discourse analysis. Results: the service users' and health personnel's discourses converged towards welcoming care practices able to build relationships of trust and lasting bonds, thus facilitating therapeutic processes that both groups characterized as producing solutions. Conclusion: the care provided to problematical drug users is progressing towards comprehensiveness, by way of user embracement practices that users and professionals describe as dialog-based, affective and potentially leading to solutions in a context favorable to autonomy and stronger citizenship.

Objetivo: analizar prácticas de acogida en la atención a la persona en uso problemático de drogas bajo el enfoque de la integralidad. Metodología: estudio cualitativo, siendo que la categoría analítica es la integralidad, realizado junto a 14 usuarios y nueve profesionales de un Centro de Atención Psicosocial en 2015, Pernambuco/Brasil. La investigación fue aprobada por el Comité de Ética de Investigación. El material empírico fue producido por medio de cuatro talleres de reflexión y analizado mediante la técnica de análisis de discurso. Resultados: los discursos de los usuarios del servicio y de profesionales convergen hacia prácticas de cuidado acogedoras, capaces de construir relaciones de confianza y vínculos consistentes, lo que facilita procesos terapéuticos caracterizados por los dos grupos como siendo resolutivos. Conclusión: El cuidado ofrecido a personas con uso problemático de drogas va hacia la integridad, con prácticas de acogida calificadas como siendo dialógicas, afectivas y con potencial para resolución en un contexto favorable a la autonomía y al fortalecimiento de la ciudadanía.

Cryptococcus gattii VGII isolated from native forest and river in Northern Brazil.

BACKGROUND: Cryptococcosis is a global invasive mycosis associated with significant morbidity and mortality. In the northern region of Brazil, this disease is caused by Cryptococcus neoformans genotype VNI and Cryptococcus gattii genotype VGII. However, few environmental studies have been conducted in this large tropical area. AIMS: This study was performed to isolate, genotype, and determine the frequency of cryptococcal agents in environmental samples near Manaus, Amazonas, Brazil. METHODS: A total of 970 environmental samples (290 from soil, 290 from decaying plants, 5 from insects, 280 from the Negro river, and 105 from small streams within the city of Manaus) were collected and plated on Niger seed agar. In addition, 20 sub-cultures obtained from each positive sample were analyzed by PCR-RFLP (URA5) and PCR for genotyping and determination of mating type. RESULTS: Six samples were positive for isolates from the C. gattii species complex. Of those, three samples were from Adolpho Ducke Forest Reserve and three were from the Negro river. All isolates were C. gattii genotype VGII (mating type MATα). CONCLUSION: Genotype VGII proved to be the most important genotype found in the environmental samples. The genotype VGII has been described as one of the most virulent and less susceptible to antifungals and responsible for important outbreaks. This is the first study to demonstrate isolation of C. gattii (VGII) from the Negro river.

Aspectos psicossociais da sexualidade na adolescência: diálogos e aprendizagem na escola / Psychosocial aspects of sexuality in adolescence: dialogue and learning in school

A sexualidade se manifesta com mais intensidade na adolescência, fase em que há transformações intensas. Na escola, faltam espaços de discussão sobre a temática. Pretendeu-se compreender os aspectos psicossociais da sexualidade de adolescentes, revelados através do desenvolvimento de oficinas. Trata-se de estudo qualitativo, baseado na pesquisa-ação e na metodologia da problematização, realizado em uma escola pública do município de Juazeiro/BA, através da oficina crítico-emancipatória "Diz aí, professor, pois quero curtir a vida!", com adolescentes do Ensino Fundamental II e Médio. Os resultados indicam que muitos adolescentes têm receio de conversar com a família por acreditarem que serão repreendidos, bem como têm pouco conhecimento sobre Doenças Sexualmente Transmissíveis e sobre direitos sexuais e reprodutivos. A homossexualidade foi o tema mais polêmico e com opiniões divergentes. Logo, percebe-se a necessidade de enfatizar a educação sexual e reprodutiva nas escolas, abordando o tema de forma alternativa ao modelo tradicional. Os tabus, as crenças e os mitos referentes à sexualidade na adolescência precisam ser discutidos por meio de estratégias que reconheçam os adolescentes como atuantes. Além disso, é importante uma aprendizagem mais reflexiva, com princípios mais humanos, no que concerne à sexualidade, diminuindo o estigma, a discriminação e a violência na sociedade (AU).

The sexuality manifests itself more strongly in adolescence, a phase were there are intense transformations. At school, there is no room for discussion on the subject. The aim of this study was to understand the psychosocial aspects of teenager's sexuality, revealed through the development of workshops. A qualitative study, based on action research and problem-solving methodology, performed in a public school in the city of Juazeiro, Bahia, through the critical-emancipatory workshop "Tell me teacher, because I want to enjoy life!" with First and Secondary School Students. The results indicate that many teenagers are afraid to talk to their family because they believe they will be reprimanded, as well as have little knowledge about Sexually Transmitted Diseases and about sexual and reproductive rights. The homosexuality was the most controversial subject and with divergent opinions. Therefore, it is necessary to emphasize sexual and reproductive education in schools, approaching the theme in an alternative way to the traditional model. The taboos, beliefs and myths need to be discussed through strategies that recognize adolescents as performers. In addition, a more reflective learning with more humane principles is important in relation to sexuality, reducing stigma, discrimination and violence in society.regarding sexuality in adolescence need to be discussed through strategies that recognize adolescents as performers. In addition, a more reflective learning with more humane principles is important in relation to sexuality, reducing stigma, discrimination and violence in society (AU).

Configurações e dinâmicas familiares de mulheres-mães durante trajetória na dependência química / Adjustments and dynamics of women-mothers during their drug dependence trajectory / Ajustes y dinámicas de las mujeres-madres de familia durante la trayectoria de la dependencia química

Introdução: O uso da droga tem afligido diretamente as relações familiares, interferindo nos laços criados, no amparo e na relação segura, adquirida ao longo da convivência familiar. Objetivo: Analisar a dinâmica familiar de mulheres-mães, antes, durante e após o convívio com as drogas. Material e método: Estudo qualitativo, exploratório e descritivo, utilizando entrevista semi-estruturada, com mulheres-mães internadas em um centro de recuperação de Petrolina-PE. Resultados: No período anterior às drogas as relações familiares eram referenciadas como boas, em oposição ao contexto às drogas, na qual evidenciou-se a naturalização da vida na rua, o roubo, a prostituição e a exploração sexual de menor, como meios de vida para sustentar o vício. A rejeição familiar esteve presente, modificando-se com a internação das depoentes, quando se apontou a possibilidade de reconstituição familiar. Conclusão: A participação da família no tratamento e cuidado das depoentes foi fundamental para fortalecer a aproximação das relações afetivas, o rompimento do estigma e preconceito tão prevalentes na sociedade. É necessário, também, que os profissionais de saúde, principalmente os enfermeiros estejam atentos e promovam a integração entre cuidado e cuidador, bem como conheçam a família e o contexto familiar em que vivem para traçar um cuidado mais efetivo.

Introduction: Drug abuse directly affects family relationships, interfering with established ties for support and safe relationships, gained over the family life. Objective: To analyze the family dynamics of women-mothers, before, during and after the interaction with drugs. Methods: A qualitative, exploratory and descriptive study using semi-structured interviews with women-mothers admitted to a Petrolina recovery center. Results: In the period previous to drug abuse, family relationships were referred to as good, as opposed to the period of drug addiction context in which naturalization of life on the street, theft, prostitution and child sexual exploitation were present as ways to enable their habit. Family rejection was present, which changed with the admission of the interviewees to the center, when the possibility of family reconstitution was addressed. Conclusion: Family participation in the treatment and care of the drug addict was essential to strengthen the approach towards affective relationships and taking away the stigma and prejudice so prevalent in society. It is also necessary that health professionals, especially nurses be aware and promote the integration of care and caregiver and know the family and the family context in which they live to implement more effective care.

Introducción: El uso de la drogas está afectando directamente las relaciones familiares, lo que interfiere con los vínculos establecidos en el apoyo y una relación segura, adquirida a lo largo de la vida familiar. Objetivo: Analizar la dinámica de las mujeres-madres de familia, antes, durante y después de la interacción con las drogas. Métodos: Estudio cualitativo, exploratorio y descriptivo mediante entrevistas semiestructuradas con las mujeres-madres ingresadas en un centro de recuperación de Petrolina. Resultados: En el período anterior a las drogas las relaciones familiares fueron referenciadas como buenas, en comparación con el contexto de drogas, en el que se presentaron la naturalización de la vida en la calle, el robo, la prostitución y la explotación sexual infantil, como sustento para apoyar su hábito. El rechazo de la familia estaba presente, cambiando con la admisión de las entrevistadas, cuando se refirió a la posibilidad de la reconstrucción familiar. Conclusión: La participación de la familia en el tratamiento y cuidado de las dependientes fue fundamental para fortalecer el enfoque de las relaciones afectivas, la ruptura del estigma y el prejuicio tan común en la sociedad. También es necesario que los profesionales de la salud, especialmente las enfermeras, estén muy atentos y promuevan la integración de la atención y el cuidador; también que conozcan la familia y el contexto familiar en el que viven para trazar un cuidado más eficaz.

Molecular typing of environmental Cryptococcus neoformans/C. gattii species complex isolates from Manaus, Amazonas, Brazil.

Cryptococcus neoformans and Cryptococcus gattii are the main causative agents of cryptococcosis, a systemic fungal disease that affects internal organs and skin, and which is acquired by inhalation of spores or encapsulated yeasts. It is currently known that the C. neoformans/C. gattii species complex has a worldwide distribution, however, some molecular types seem to prevail in certain regions. Few environmental studies of Cryptococcus have been conducted in the Brazilian Amazon. This is the first ecological study of the pathogenic fungi C. neoformans/C. gattii species complex in the urban area of Manaus, Amazonas, Brazil. A total of 506 samples from pigeon droppings (n = 191), captive bird droppings (n = 60) and tree hollows (n = 255) were collected from June 2012 to January 2014 at schools and public buildings, squares, pet shops, households, the zoo and the bus station. Samples were plated on niger seed agar (NSA) medium supplemented with chloramphenicol and incubated at 25°C for 5 days. Dark-brown colonies were isolated and tested for thermotolerance at 37°C, cycloheximide resistance and growth on canavanine-glycine-bromothymol blue agar. Molecular typing was done by PCR-RFLP. Susceptibility to the antifungal drugs amphotericin B, fluconazole, itraconazole and ketoconazole was tested using Etest(®) strips. In total, 13 positive samples were obtained: one tree hollow (C. gattiiVGII), nine pigeon droppings (C. neoformansVNI) and three captive bird droppings (C. neoformansVNI). The environmental cryptococcal isolates found in this study were of the same molecular types as those responsible for infections in Manaus.

Uso de substâncias psicoativas por estudantes rurais: distribuição por sexo e idade / Consumo de sustancias psicoactivas en una escuela rural: distribución por sexo y edad / Use of psychoactive substances by students of a rural school: distribution by sex and age

Objetivo: analisar o uso na vida de álcool, tabaco e outras drogas e sua distribuição por sexo e idade em estudantes do fundamental II e médio, de uma escola pública e rural. Método: estudo quantitativo, descritivo e transversal, realizado de agosto a novembro de 2011 com 146 estudantes de Petrolina e analisado pelo programa Microsoft Excel 2007. Como critérios de elegibilidade elegeu-se ser maior de 18 anos e assinar o Termo de Consentimento Livre e Esclarecido ou, se menor, obter a assinatura do responsável. Resultados: verificou-se maior uso na vida por álcool, predominando mulheres de 16 a 18 anos; 16,44% usaram cigarro, sem distinção do sexo; e 3,42% usaram drogas ilícitas, prevalecendo os homens. Conclusões: estratégias educacionais, através da escola, família ou governo, são necessárias na promoção de diálogos com os alunos, reduzindo o uso de álcool, tabaco e outras drogas entre estudantes.

Objective: to analyze the lifetime use of alcohol, tobacco and other drugs, and their distribution by sex and age of students of elementary and high school, in a rural public school. Method: quantitative, descriptive and cross-sectional study, conducted from August to November of 2011 with 146 students in Petrolina and analyzed by Microsoft Excel 2007. As for the criteria of eligibility, elligible students had to be over 18 and sign the Informed Consent or, if underage, obtain the parents’ signature. Results: there was greater lifetime use of alcohol, predominantly among women of 16-18 years old; 16.44% used cigarettes, without distinction of sex; and 3.42% used illicit drugs, prevailing men. Conclusions: educational strategies, through school, family or government, are necessary to promote dialogues with students, reducing the use of alcohol, tobacco and other drugs among students.

Objetivo: analizar el consumo de alcohol, tabaco y otras drogas y su distribución por sexo y edad entre estudiantes de una escuela pública rural de primaria y secundaria. Método: estudio cuantitativo, descriptivo y transversal, realizado entre agosto y noviembre de 2011 con 146 estudiantes en Petrolina (PE, Brasil) y analizados com Microsoft Excel 2007. Como criterios de elegibilidad se exigió ser mayor de 18 años yFirmar un consentimiento informado o, para menores de edad, obtener la firma del responsable. Resultados: se verificó mayor consumo de alcohol, predominantemente entre mujeres de 16-18 años; 16,44% consumen cigarrillos, sin distinción de sexo; y 3,42% consumen drogas ilícitas, prevaleciendo los hombres. Conclusiones: estratégias educativas, a través de la escuela, la familia o el gobierno, son necesarios en la promoción de diálogos con los estudiantes, para reducir el consumo de alcohol, tabaco y otras drogas entre los estudiantes.

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)

Caracterización de aislamientos clínicos del complejo Cryptococcus neoformans-Cryptococcus gattii, del Estado Amazonas - Brasil / Characterization of clinical isolates of the Cryptococcus neoformans-Cryptococcus gattii species complex from the Amazonas State in Brazil

Antecedentes. La diferenciación y clasificación de las especies patógenas del género Cryptococcus aporta datos importantes para la asistencia clínica y para estudios epidemiológicos. Objetivos. El objetivo de este trabajo fue caracterizar 40 aislamientos clínicos del complejo Cryptococcus neoformans de pacientes que fueron atendidos en la Fundación de Medicina Tropical de Amazonas desde 2006 hasta 2008. Métodos. Se utilizaron métodos fenotípicos (producción de enzimas y pruebas de sensibilidad a los antifúngicos) y moleculares (URA5-RFLP). Resultados. Los pacientes con VIH/sida fueron los más afectados de criptococosis. Se observó que 31 (75,5%) y 9 (22,5%) de los aislamientos fueron Cryptococcus neoformans y Cryptococcus gattii, respectivamente. La producción de proteasa y fosfolipasa fue alta en la mayoría de las cepas. Utilizando la prueba de difusión en disco (CLSI M44-A) se observó que el 81, 35 y 100% de los aislamientos de C. neoformans fueron sensibles al fluconazol, itraconazol y amphotericin B, respectivamente, mientras que 78, 56 y 100% de los aislamientos de C. gattii fueron sensibles a estas sustancias. El valor promedio de la concentración mínima inhibitoria (CMI) para C. neoformans y C. gattii fue de 0,26 y 0,58mg/ml, respectivamente. Todos los aislamientos (9) de C. gattii presentaron un patrón de electroforesis compatible con el genotipo VGII, y todos los aislamientos (31) de C. neoformans presentaron el genotipo VNI. Conclusiones. Este estudio confirma la importancia del HIV/sida para la epidemiología de la criptococosis, la sensibilidad de los aislamientos a la anfotericina B y la alta prevalencia de los genotipos moleculares VNI y VGII en el norte de Brasil(AU)

Background. The differentiation and classification of pathogenic Cryptococcus species provides useful data for epidemiological studies and for the clinical diagnosis and treatment of patients. Aims. The aim of this study was to characterise 40 clinical Cryptococcus isolates obtained from patients at the Tropical Medicine Foundation of Amazonas (FMTAM) from 2006 to 2008. Methods. It was used phenotypic (i.e., enzyme production and antifungal resistance) and molecular biological (URA5-RFLP) experiments. Results. Patients with HIV/AIDS were most affected with cryptococcosis. Thirty-one (75.5%) of the clinical isolates were classified as Cryptococcus neoformans and 9 (22.5%) as Cryptococcus gattii. High amounts of protease and phospholipase enzymes were produced by most of the isolates. Using the disk diffusion test (CLSI M44-A), 81, 35 and 100% of the C. neoformans isolates were characterized as susceptible to fluconazole, itraconazole and amphotericin B, respectively, whereas 78, 56 and 100% of the C. gattii isolates were susceptible to these antimicrobial agents. The average of Minimal Inhibitory Concentration (MIC) for C. neoformans and C. gattii isolates was 0.26 and 0.58µg/mL, respectively. The 9 isolates of C. gattii had a fingerprint pattern comparable with the VGII molecular type, while all 31 isolates of C. neoformans presented with a pattern consistent with the VNI type. Conclusions. This study confirms the importance of HIV/AIDS for the cryptococcosis epidemiology, the susceptibility of the isolates to amphotericin B and the high prevalence of the molecular genotypes VNI and VGII in the north of Brazil(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.

Characterization of clinical isolates of the Cryptococcus neoformans-Cryptococcus gattii species complex from the Amazonas State in Brazil.

BACKGROUND: The differentiation and classification of pathogenic Cryptococcus species provides useful data for epidemiological studies and for the clinical diagnosis and treatment of patients. AIMS: The aim of this study was to characterise 40 clinical Cryptococcus isolates obtained from patients at the Tropical Medicine Foundation of Amazonas (FMTAM) from 2006 to 2008. METHODS: It was used phenotypic (i.e., enzyme production and antifungal resistance) and molecular biological (URA5-RFLP) experiments. RESULTS: Patients with HIV/AIDS were most affected with cryptococcosis. Thirty-one (75.5%) of the clinical isolates were classified as Cryptococcus neoformans and 9 (22.5%) as Cryptococcus gattii. High amounts of protease and phospholipase enzymes were produced by most of the isolates. Using the disk diffusion test (CLSI M44-A), 81, 35 and 100% of the C. neoformans isolates were characterized as susceptible to fluconazole, itraconazole and amphotericin B, respectively, whereas 78, 56 and 100% of the C. gattii isolates were susceptible to these antimicrobial agents. The average of Minimal Inhibitory Concentration (MIC) for C. neoformans and C. gattii isolates was 0.26 and 0.58 µg/mL, respectively. The 9 isolates of C. gattii had a fingerprint pattern comparable with the VGII molecular type, while all 31 isolates of C. neoformans presented with a pattern consistent with the VNI type. CONCLUSIONS: This study confirms the importance of HIV/AIDS for the cryptococcosis epidemiology, the susceptibility of the isolates to amphotericin B and the high prevalence of the molecular genotypes VNI and VGII in the north of Brazil.

Selection and optimization of PCR-based methods for the detection of Histoplasma capsulatum var. capsulatum.

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.