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Introduction. A specialized service for antifungal blood level determination is not available in Colombia. This service is essential for the proper follow-up of antifungal therapies. Objective. To standardize and validate a simple, sensitive, and specific protocol based on high-performance liquid chromatography with a diode array detector for voriconazole blood level quantification. Materials and methods. We used an Agilent HPLC™ series-1200 equipment with a UVdiode array detector with an analytical column Eclipse XDB-C18 and pre-column Eclipse- XDB-C18 (Agilent). We used voriconazole as the primary control and posaconazole as an internal control. We performed the validation following the Food and Drug Administration (FDA) recommendations. Results. The best chromatographic conditions were: Column temperature of 25°C, UV variable wavelength detection at 256 nm for voriconazole and 261 nm for posaconazole (internal standard); 50 μl of injection volume, 0,8 ml/min volume flow, 10 minutes of run time, and mobile phase of acetonitrile:water (60:40). Finally, retention times were 3.13 for voriconazole and 5.16 minutes for posaconazole. Quantification range varied from 0.125 μg/ml to 16 μg/ml. Conclusion. The selectivity and chromatographic purity of the obtained signal, the detection limits, and the standardized quantification make this method an excellent tool for the therapeutic monitoring of patients treated with voriconazole.

Introduction: Fusarium is a very heterogeneous group of fungi, difficult to classify, with a wide range of living styles, acting as saprophytes, parasites of plants, or pathogens for humans and animals. Prevalence of clinical fusariosis and lack of effective treatments have increased the interest in the precise diagnosis, which implies a molecular characterization of Fusarium populations. Objective: We compared different genotyping markers in their assessment of the genetic variability and molecular identification of clinical isolates of Fusarium. Materials and methods: We evaluated the performance of the fingerprinting produced by two random primers: M13, which amplifies a minisatellite sequence, and (GACA)4, which corresponds to a simple repetitive DNA sequence. Using the Hunter Gaston Discriminatory Index (HGDI), an analysis of molecular variance (AMOVA), and a Mantel test, the resolution of these markers was compared to the reference sequencing-based and PCR genotyping methods. Results: The highest HGDI value was associated with the M13 marker followed by (GACA)4. AMOVA and the Mantel tests supported a strong correlation between the M13 classification and the reference method given by the partial sequencing of the transcription elongation factor 1-alpha (TEF1-α) and rDNA 28S. Conclusion: The strong correlation between the M13 classification and the sequencingbased reference together with its higher resolution demonstrates its adequacy for the characterization of Fusarium populations.

Invasive fungal diseases (IFD) contribute significantly to worldwide morbidity and mortality, but their frequency is not well-described in some countries. The present work describes the frequency of IFD in a specialized laboratory in Colombia. A retrospective, descriptive study was implemented between March 2009 and December 2015. Results: 13,071 patients with clinical suspicion of IFD were referred during the study period, from which 33,516 biological samples were processed and analyzed using 14 laboratory methods. Diagnosis was confirmed in 1425 patients (11%), distributed according to the mycoses of interest analyzed here: histoplasmosis in 641/11,756 patients (6%), aspergillosis in 331/10,985 patients (3%), cryptococcosis in 239/8172 patients (3%), pneumocystosis in 111/1651 patients (7%), paracoccidioidomycosis in 60/10,178 patients (0.6%), and invasive candidiasis in 48/7525 patients (0.6%). From the first year of the study period to the last year, there was a 53% increase in the number of cases of IFD diagnosed. Our laboratory experienced a high frequency of IFD diagnosis, possibly attributable to the availability of a greater range of diagnostic tools. Frequency of IFD in this study was atypical compared with other studies, probably as a result of the single laboratory-site analysis. This demonstrates that implementing educational strategies helps to create a high index of clinical suspicion, while the availability and utilization of appropriate diagnostic assays assure greater reliability in identification of these cases.

Iron is an essential growth element of virtually all microorganisms and its restriction is one of the mechanisms used by macrophages to control microbial multiplication. Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis, an important systemic mycosis in Latin America, is inhibited in its conidia-to-yeast conversion in the absence of iron. We studied the participation of iron in the nitric oxide (NO)-mediated fungicidal mechanism against conidia. Peritoneal murine macrophages activated with 50U/mL of IFN-gamma or treated with 35 µM Deferoxamine (DEX) and infected with P. brasiliensis conidia, were co-cultured and incubated for 96 h in the presence of different concentrations of holotransferrin (HOLO) and FeS0(4). The supernatants were withdrawn in order to assess NO2 production by the Griess method. The monolayers were fixed, stained and observed microscopically. The percentage of the conidia-to-yeast transition was estimated by counting 200 intracellular propagules. IFN-gamma-activated or DEX-treated Mthetas presented marked inhibition of the conidia-to-yeast conversion (19 and 56%, respectively) in comparison with non-activated or untreated Mthetas (80%). IFN-gamma-activated macrophages produced high NO levels in comparison with the controls. Additionally, when the activated or treated-macrophages were supplemented with iron donors (HOLO or FeSO4), the inhibitory action was reversed, although NO production remained intact. These results suggest that the NO-mediated fungicidal mechanism exerted by IFN-gamma-activated macrophages against P. brasiliensis conidia, is dependent of an iron interaction.O ferro é elemento essencial para o crescimento de microrganismos e sua limitação é um dos mecanismos usados por macrófagos para controlar a multiplicação microbiana. Paracoccidioides brasiliensis, o agente da paracoccidioidomicose, uma das micoses sistêmicas mais importantes na América Latina, é inibido em sua conversão de conídia-à-levedura na ausência do ferro. Estudamos a participação do ferro no mecanismo fungicida mediado pelo óxido nítrico (NO) na sua interação com as conídias do fungo. Macrófagos peritoneais murinos ativados com 50U/mL de IFN-gama ou tratados com 35 µM Deferoxamina (DEX) e infectados com conídias do P. brasiliensis foram co-cultivados e incubados por 96 h na presença de concentrações diferentes de holotransferrina (HOLO) e FeS0(4). Os sobrenadantes foram retirados a fim de avaliar a produção de NO2 pelo método de Griess. Os macrófagos eram fixados, corados e observados ao microscópio. A porcentagem da transição de conídia-à-levedura foi estimada contando 200 propágulos intracelulares. Os macrófagos ativados com citocina ou tratados com DEX apresentaram inibição marcada da conversão de conídia-à-levedura (19 e 56%, respectivamente) em comparação com macrófagos controle (80%). Os macrófagos ativados com IFN-gama produziram elevação nos níveis de NO em comparação com macrófagos não-tratados ou não-activados. Adicionalmente, quando as monocapas ativadas ou tratadas foram suplementadas com doadores do ferro (HOLO ou FeSO4), a ação inibitória foi revertida embora a produção de NO permanecesse intacto. Estes resultados sugerem que o mecanismo fungicida mediado pelo NO exercido por macrófagos ativados com IFN-gama contra conídias do P. brasiliensis é dependente de uma interação do ferro.

La paracoccidioidomicosis, micosis profunda de gran importancia en Latinoamérica, se caracteriza por presentar una respuesta inflamatoria aguda en el pulmón, órgano blanco del proceso infeccioso. En las etapas iniciales, esta respuesta se caracteriza principalmente por un infiltrado de polimorfonucleares neutrófilos (PMN), los que, de acuerdo con estudios histopatológicos y de lavados broncoalveolares, representan más del 85% de las células allí presentes. Las evidencias presentadas en esta revisión sugieren que el PMN participa activamente en la respuesta inflamatoria aguda inducida por Paracoccidioides brasiliensis, como lo demuestra la inhibición de su crecimiento después de su interacción con tales PMN. Igualmente, se conoce que algunas etapas del mecanismo fungicida exhibido por esta célula fagocítica están mediadas por los reactivos intermediarios del oxígeno, así como también por el efecto activador de algunas citocinas como IFN y, GM-CSF e IL-lB. En efecto, fracciones o componentes del hongo inducen la migración al sitio inflamatorio y la activación de los PMN, evidenciada por el aumento en los productos del estallido respiratorio.

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.

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.

Se describen los procedimientos de purificación empleados para la separación de las fracciones antigénicas a partir de un material somático obtenido por rotura de células levaduras completas de P. brasiliensis. Dichas fracciones mostraron ser proteínas con pesos moleculares de 66 y 85 Kd; la primera de ellas reaccionó con sueros específicos produciendo una banda de precipitado idéntica a una de las 3 desarrolladas por el antígeno total. Los resultados señalan la posibilidad de obtener antígenos purificados, químicamente identificados y cuyo uso pudiera, en el futuro, representar ventajas para el diagnóstico serológico de la paracoccidioidomicosis, permitiendo separar, repetidamente, solo aquel componente reconocidamente activo.

Resumen Introducción: En la literatura colombiana son escasos los reportes acerca de la epidemiología de la tinea capitis. Objetivo : Realizar un estudio retrospectivo para describir el comportamiento de esta micosis y de sus agentes etiológicos, en una serie de pacientes remitidos a un centro de diagnóstico especializado en Medellín, Colombia. Métodos : Estudio retrospectivo donde se analizaron los registros de pacientes remitidos entre los años 1994 y 2013 para estudio micológico a la Unidad de Micología Médica y Experimental de la Corporación para Investigaciones Biológicas (CIB), en Medellín, Colombia. Resultados : Fueron analizados 415 pacientes con sospecha clínica de tinea capitis, 133 (32%) de los cuales fueron confirmados por el laboratorio. La mayoría de los pacientes positivos, 124/133 (93%), fueron menores de edad y 89/133 (67%) correspondieron al sexo masculino. En 52 de los 133 casos comprobados se pudo determinar algún factor de riesgo asociado: el contacto con animales fue el principal factor de riesgo en 39/52 pacientes (75%). El examen directo fue positivo en el 87% y el cultivo para hongos en el 92% de los casos comprobados. El agente etiológico más frecuentemente aislado fue Microsporum canis (86%), seguido con una amplia diferencia por Microsporum gypseum(4%), Trichophyton tonsurans (3%), Trichophyton mentagrophytes (3%), Microsporum audouinii (3%) y Microsporum spp. (1%). Conclusión : Nuestros resultados representan una casuística importante para la epidemiología de la tinea capitis en Colombia. En ausencia de estudios más extensos en cobertura geográfica y en población estudiada que permitan conocer la incidencia real de esta micosis en nuestro medio, estos datos deben ser considerados como aporte valioso en el conocimiento de los agentes etiológicos de tinea capitis más frecuentes en el país.