Resumen Introducción: La clasificación a nivel de especies de las levaduras del género Candida de origen clínico es fundamental para el diagnóstico y la instauración de un adecuado tratamiento para el paciente. Se realizó un estudio de concordancia de cinco metodologías usadas para la identificación de aislamientos orales de Candida spp en Colombia. Métodos: Sesenta y siete aislamientos de Candida spp fueron identificados a nivel de especie utilizando; API® 20 C AUX‚ Vitek® 2 Compact, MALDI TOF (Vitek® MS y Microflex®) y una prueba molecular, PCR Panfungal y secuenciación. Un análisis del costo comercial y tiempo de procesamiento de las muestras por cada método fue realizado mediante el análisis gráfico de ambas variables. Resultados: La PCR Panfungal y secuenciación diferenció 12 especies de Candida‚ los métodos Vitek® MS y Microflex® identificaron 9 especies y los métodos API® 20 C AUX y Vitek® 2 Compact identificaron 8 especies. El análisis de Kappa ponderado (wK) demostró una concordancia alta entre los métodos PCR Panfungal y secuenciación‚ Vitek® MS‚ Microflex® y API® 20 C AUX‚ concordancias agrupadas en las categorías buena y muy buena (wK 0.62 - 0.93); los Kp que involucraron el método Vitek® 2 Compact presentaron concordancias moderadas o buenas frente a los otros métodos (wK 0.56 - 0.73). Las metodologías basadas en MALDI TOF MS requirieron 4 minutos para generar un resultado y el método Microflex® fue el método que en nuestro medio presentó el menor precio de venta del servicio. Conclusión: Los métodos evaluados presentaron una alta concordancia en sus resultados‚ siendo más alta para los métodos moleculares y las metodologías basadas en MALDI TOF MS; estas últimas son metodologías más rápidas, económicas y precisas, las cuales se presentan como alternativas prometedoras para la identificación rutinaria de especies de levaduras del género Candida.

Abstract Background: The yeasts species determination is fundamental not only for an accurate diagnosis but also for establishing a suitable patient treatment. We performed a concordance study of five methodologies for the species identification of oral isolates of Candida in Colombia. Methods: Sixty-seven Candida isolates were tested by; API® 20C-AUX, Vitek®2 Compact, Vitek®MS, Microflex® and a molecular test (panfungal PCR and sequencing). The commercial cost and processing time of the samples was done by graphical analysis. Results: Panfungal PCR differentiated 12 species of Candida, Vitek®MS and Microflex® methods identified 9 species, and API® 20C-AUX and Vitek®2 Compact methods identified 8 species each. Weighted Kappa (wK) showed a high agreement between Panfungal PCR, Vitek®MS, Microflex® and API® 20C-AUX (wK 0.62-0.93). The wK that involved the Vitek®2 Compact method presented moderate or good concordances compared with the other methods (wK 0.56-0.73). Methodologies based on MALDI TOF MS required 4 minutes to generate results and the Microflex® method had the lowest selling price. Conclusion: The methods evaluated showed high concordance in their results, being higher for the molecular methods and the methodologies based on MALDI TOF. The latter are faster and cheaper, presenting as promising alternatives for the routine identification of yeast species of the genus Candida.

ABSTRACT Objective The need to build public health systems that ensure comprehensive child development enables a characterization of the factors that favor or restrict the integral development of children under the age of six. That is in order to identify them as necessary components to be taken into account in public policies. Method A survey was carried out with 1 177 mothers or family caregivers of children under six years old in 16 municipalities of Cundinamarca and Boyacá, and the respective socio-cognitive development of their own sons and daughters was measured with the Haizea-Llevant table and also tasks related to the use of core knowledge systems. Results Through bivariate and multivariate statistical analyzes, it was found that the factors that are significant to address a public health proposal that seeks the integral development of children under six years of age are: socio-economic conditions of households, complications in childbirth, the starting age of solid foods, mothers' working shifts, rules on daily routines and play practices such as reading, painting and sport activities. Conclusions A health system that acknowledges the results presented should offer specialized care that seeks welfare in childhood and early childhood, which can only be achieved if health policies begin to consider domestic and daily factors that cannot be excluded from public policies and it should guarantee different levels of intervention of social and particular impact.

RESUMEN Objetivo La necesidad de construir sistemas de salud pública que garanticen el desarrollo infantil integral, permite realizar una caracterización de los factores que favorecen o limitan el desarrollo integral de niñas y niños menores de seis años, para identificarlos como componentes necesarios a ser tenidos en cuenta en políticas públicas. Método Se realizó una encuesta a 1 177 madres o cuidadores de niños menores de seis años en 16 municipios de Cundinamarca y Boyacá, y se midió el respectivo desarrollo socio-cognitivo de los menores participantes con la tabla Haizea-Llevant y tareas relacionadas con el uso de sistemas de conocimiento intra-específicos. Resultados Mediante análisis estadísticos bivariados y multivariados se encontró que los factores que resultan significativos para atender a una propuesta de salud pública que busque el desarrollo integral de los niños menores de seis años son: las condiciones socio-económicas de los hogares, las complicaciones en el parto, la edad de inicio de alimentos sólidos, los tiempos de trabajo de las madres, la reglas sobre rutinas diarias y las prácticas de juego: lectura, pintura y actividades deportivas. Conclusiones Un sistema de salud que reconozca los resultados presentados, debería ofrecer atención especializada que procure el bienestar en la infancia y la niñez temprana, lo cual solo puede lograrse si las políticas de salud comienzan a considerar factores de orden doméstico y cotidiano que no pueden estar por fuera de políticas públicas, asegurando diversos niveles de intervención, impacto social y particular.

RESUMEN Objetivo La necesidad de construir sistemas de salud pública que garanticen el desarrollo infantil integral, permite realizar una caracterización de los factores que favorecen o limitan el desarrollo integral de niñas y niños menores de seis años, para identificarlos como componentes necesarios a ser tenidos en cuenta en políticas públicas. Método Se realizó una encuesta a 1 177 madres o cuidadores de niños menores de seis años en 16 municipios de Cundinamarca y Boyacá, y se midió el respectivo desarrollo socio-cognitivo de los menores participantes con la tabla Haizea-Llevant y tareas relacionadas con el uso de sistemas de conocimiento intra-específicos. Resultados Mediante análisis estadísticos bivariados y multivariados se encontró que los factores que resultan significativos para atender a una propuesta de salud pública que busque el desarrollo integral de los niños menores de seis años son: las condiciones socio-económicas de los hogares, las complicaciones en el parto, la edad de inicio de alimentos sólidos, los tiempos de trabajo de las madres, la reglas sobre rutinas diarias y las prácticas de juego: lectura, pintura y actividades deportivas. Conclusiones Un sistema de salud que reconozca los resultados presentados, debería ofrecer atención especializada que procure el bienestar en la infancia y la niñez temprana, lo cual solo puede lograrse si las políticas de salud comienzan a considerar factores de orden doméstico y cotidiano que no pueden estar por fuera de políticas públicas, asegurando diversos niveles de intervención, impacto social y particular.

ABSTRACT Objective The need to build public health systems that ensure comprehensive child development enables a characterization of the factors that favor or restrict the integral development of children under the age of six. That is in order to identify them as necessary components to be taken into account in public policies. Method A survey was carried out with 1 177 mothers or family caregivers of children under six years old in 16 municipalities of Cundinamarca and Boyacá, and the respective socio-cognitive development of their own sons and daughters was measured with the Haizea-Llevant table and also tasks related to the use of core knowledge systems. Results Through bivariate and multivariate statistical analyzes, it was found that the factors that are significant to address a public health proposal that seeks the integral development of children under six years of age are: socio-economic conditions of households, complications in childbirth, the starting age of solid foods, mothers' working shifts, rules on daily routines and play practices such as reading, painting and sport activities. Conclusions A health system that acknowledges the results presented should offer specialized care that seeks welfare in childhood and early childhood, which can only be achieved if health policies begin to consider domestic and daily factors that cannot be excluded from public policies and it should guarantee different levels of intervention of social and particular impact.

La histoplasmosis, micosis sistémica y endémica en una amplia zona de las Américas, es causada por el hongo dimórfico Histoplasma capsulatum. Tradicionalmente, el diagnóstico de esta entidad se realiza por métodos microbiológicos directos y biopsias que emplean una variedad de coloraciones especiales tales como Wright, Giemsa y plata metenamina, entre otras, así como por cultivo; este último representa el estándar de oro. Se emplean, igualmente, métodos indirectos que incluyen la detección de anticuerpos y antígenos. Los valores de sensibilidad y especificidad en ambos métodos son variables, y los resultados dependen, a su vez, de la forma clínica de la enfermedad que presente el paciente y de su estado inmune. Recientemente, la biología molecular ha permitido introducir nuevas herramientas que han sido utilizadas para la detección e identificación de H.capsulatum, una de ellas es la PCR anidada que se caracteriza por sus altos niveles de sensibilidad y especificidad. De igual forma, estas técnicas moleculares han permitido realizar análisis evolutivos, estudios de diversidad genética y un sinnúmero de estudios de epidemiología molecular, a partir de los cuales se ha logrado recopilar información valiosa sobre la variabilidad genética de este microorganismo. En esta revisión se describen los métodos de laboratorio convencionales y las técnicas moleculares más empleadas para el diagnóstico de la histoplasmosis; así como también algunas de sus aplicaciones en la epidemiología y biología molecular de este hongo.

Histoplasma capsulatum is the causative agent of histoplasmosis, a systemic and endemic mycosis widely distributed in the Americas. Diagnosis of histoplasmosis is traditionally accomplished by means of direct preparations and biopsies stained by especial methods, as well as by isolation of fungus in culture; the latter is considered the gold standard. Indirect methods, including immunological tests to detect antibodies and/or antigens, are also valuable; both direct and indirect methods present sensitivity and specificity ranges that vary depending on the clinical form of the disease and the immune status of the host. Recently, molecular biology has allowed implementing new tools to detect and identify H. capsulatum, and several molecular tests, such as nested-PCR, are being used for the diagnosis of histoplasmosis, and so provide high sensitivity and specificity values. In addition, these molecular techniques have made it possible to perform evolution analysis, genetic diversity research, and molecular epidemiology, thus compiling valuable information on the genetic variability of this microorganism. In this review, the conventional and molecular methods employed for the diagnosis of histoplasmosis have been described, as have some of the applications of these molecular techniques to this fungal pathogen's epidemiology and molecular biology.

Com a finalidade de determinar o papel da lisozima, um peptídeo antimicrobiano que pertence ao sistema imune inato, contra o fungo dimórfico Paracoccidioides brasiliensis, foram feitas co-culturas de uma linha de macrófagos alveolares murinos (MH-S) com as conídias do fungo na presença ou não do TNF-α e/ou um inibidor da lisozima; também foram feitos ensaios que avaliaram o efeito das concentrações fisiológicas e inflamatórias de lisozima diretamente sobre o ciclo de vida do fungo. Observamos que os macrófagos ativados com a citoquina tiveram um efeito significativo na inibição da transição conídia/levedura (p = 0,0043) e exerceram um efeito fungicida importante (p = 0,0044), matando mais de 27% das propágulas do fungo em comparação com os macrófagos não ativados. No entanto, após ser o inibidor seletivo da lisozima adicionado, o efeito fungicida foi revertido. Quando os propágulos do fungo foram expostos diretamente a diferentes concentrações da lisozima, um duplo efeito foi observado. Assim, as concentrações fisiológicas da enzima facilitaram o processo de transição conídia-levedura (p < 0,05). Contrariamente, as concentrações inflamatórias prejudicaram a transição fúngica (p < 0,0001). Quando as leveduras foram expostas a qualquer concentração de lisozima, sua capacidade de multi-brotação foi gravemente prejudicada (p < 0,0001). Além disso, mudanças ultra-estruturais, como a sub degradação, a fusão dos vacúolos dos lípidos, estruturas lamelares e interrupção da camada fibrilar foram observadas em conídios expostos à lisozima. Estes resultados sugerem que a lisozima poderia exercer um duplo papel no mecanismo antifúngico contra P. brasiliensis.

In order to determine the role of lysozyme, an antimicrobial peptide belonging to the innate immune system, against the dimorphic fungus Paracoccidioides brasiliensis, co-cultures of the MH-S murine alveolar macrophages cell line with P. brasiliensis conidia were done; assays to evaluate the effect of physiological and inflammatory concentrations of lysozyme directly on the fungus life cycle were also undertaken. We observed that TNF-α-activated macrophages significantly inhibited the conidia to yeast transition (p = 0.0043) and exerted an important fungicidal effect (p = 0.0044), killing 27% more fungal propagules in comparison with controls. Nonetheless, after adding a selective inhibitor of lysozyme, the fungicidal effect was reverted. When P. brasiliensis propagules were exposed directly to different concentrations of lysozyme, a dual effect was observed. Physiologic concentrations of the enzyme facilitated the conidia-to-yeast transition process (p < 0.05). On the contrary, inflammatory concentrations impaired the normal temperature-dependant fungal transition (p < 0.0001). When yeast cells were exposed to lysozyme, irrespective of concentration, the multiple-budding ability was badly impaired (p < 0.0001). In addition, ultra-structural changes such as subcellular degradation, fusion of lipid vacuoles, lamellar structures and interruption of the fibrilar layer were observed in lysozyme exposed conidia. These results suggest that lysozyme appears to exert a dual role as part of the anti-P. brasiliensis defense mechanisms.

Pacientes com paracoccidioidomicose apresentam, algumas vezes, fibrose pulmonar e exibem limitações respiratórias importantes. Baseados num modelo animal já estabelecido da micose, estudamos a possível contribuição de propágulos viáveis e não viáveis do Paracoccidioides brasiliensis ao desenvolvimento da fibrose. Assim, camundongos BALB/c, machos de 4 a 6 semanas de idade, foram inoculados intranasalmente com 4 x 10(6) conídios viáveis (Grupo I), ou com 6,5 x 10(6) fragmentos não viáveis de células leveduriformes (Grupo II). Animais controles (Grupo III) receberam unicamente PBS. Seis camundongos por período foram sacrificados 24, 48, 72h (inicial) e 1, 2, 4, 8, 12 e 16 semanas pós-inoculação (tardio). Os pulmões dos animais foram fixados, incluidos em parafina, cortados e corados com H & E, Tricrômico (Masson), reticulina e Grocott. Durante o período inicial houve afluxo importante de PMNs em ambos os grupos I e II, e a inflamação aguda comprometeu entre 34 a 45% dos pulmões. Depois, foram as células mononucleares as que predominaram. No grupo I, a inflamação progrediu e formaram-se granulomas os quais, às 12 semanas, ficaram confluentes e frouxos. Adicionalmente, se observaram fibras de colágeno tipo I muito densas em 66,6% e 83,3% dos animais após 8 e 12 semanas, respectivamente. As fibras do colágeno tipo III foram observadas nos animais a partir das 4 semanas pós-infecção, e 83% deles exibiram, às 12 semanas, alterações na sua distribuição e organização. Nos animais do grupo II o padrão foi diferente, pois mostraram diminuição gradual no número de focos inflamatórios e não houve formação dos granulomas. Embora animais deste grupo tivessem no período inicial pequenas alterações nas fibras de colágeno tipo I, estas desapareceram por volta da 4a semana. Os resultados indicam que a resposta tissular aos fragmentos de leveduras foi transitória, enquanto que os conídios induzem resposta inflamatória progressiva permitindo a formação de granuloma e um excesso na produção e desorganização dos colágenos I e III, permitindo finalmente a fibrose.

Patients with paracoccidioidomycosis often present pulmonary fibrosis and exhibit important respiratory limitations. Based on an already established animal model, the contribution of viable and non-viable P. brasiliensis propagules to the development of fibrosis was investigated. BALB/c male mice, 4-6 weeks old were inoculated intranasally either with 4x10(6 )viable conidia (Group I), or 6.5x10(6) fragmented yeast cells (Group II). Control animals received PBS. Six mice per period were sacrificed at 24, 48, 72h (initial) and 1, 2, 4, 8, 12 and 16 weeks post-challenge (late). Paraffin embedded lungs were sectioned and stained with H&E, trichromic (Masson), reticulin and Grocott´s. During the initial period PMNs influx was important in both groups and acute inflammation involving 34% to 45% of the lungs was noticed. Later on, mononuclear cells predominated. In group I, the inflammation progressed and granulomas were formed and by the 12th week they fussed and became loose. Thick collagen I fibers were observed in 66.6% and 83.3% of the animals at 8 and 12 weeks, respectively. Collagen III, thick fibers became apparent in some animals at 4weeks and by 12 weeks, 83% of them exhibited alterations in the organization and thickness of these elements. In group II mice, this pattern was different with stepwise decrease in the number of inflammatory foci and lack of granulomas. Although initially most animals in this group had minor alterations in thin collagen I fibers, they disappeared by the 4th week. Results indicate that tissue response to fragmented yeast cells was transitory while viable conidia evoked a progressive inflammatory reaction leading to granuloma formation and to excess production and/or disarrangement of collagens I and III; the latter led to fibrosis.