Standardization and validation of a high-efficiency liquid chromatography with a diode-array detector (HPLC-DAD) for voriconazole blood level determination. / Estandarización y validación de un método de cromatografía líquida de alta eficiencia con detector de arreglo de diodos (HPLC-DAD) para la determinación de niveles sanguíneos de voriconazol

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.

Introducción. Hasta la fecha, Colombia no cuenta con un servicio especializado de medición de niveles séricos de antifúngicos, procedimiento esencial para el adecuado seguimiento del tratamiento de infecciones fúngicas invasoras. Objetivo. Estandarizar y validar un protocolo ­simple, sensible y específico­ basado en la aplicación de cromatografía líquida de alta eficiencia acoplada con un detector de arreglo de diodos para la cuantificación de los niveles séricos de voriconazol. Materiales y métodos. Se usó un equipo HPLC-Agilent™, serie-1200, con un detector UVDAD, una columna analítica Eclipse-XDB-C18 y una pre-columna Eclipse-XDB-C18, ambas de la marca Agilent. Como control primario se utilizó voriconazol y como control interno, posaconazol. La validación se hizo cumpliendo todos los criterios de aceptación recomendados por la Food and Drug Administration (FDA). Resultados. Las mejores condiciones cromatográficas se obtuvieron con los siguientes parámetros: temperatura de la columna de 25 °C, detección UV-VWD de 261 nm, volumen de inyección de 50 µl, flujo de 0,8 ml/minuto y un tiempo de corrido de 10 minutos. La fase móvil usada fue acetonitrilo:agua (60:40) y los tiempos finales de retención fueron de 3,13 para voriconazol y de 5,16 minutos para posaconazol. El rango de cuantificación fue desde 0,125 µg/ml hasta 16 µg/ml. Conclusiones. La selectividad y la pureza de la señal cromatográfica, así como los límites de detección y cuantificación estandarizados hacen de esta metodología una excelente herramienta para el seguimiento terapéutico de pacientes tratados con voriconazol o en profilaxis con este fármaco.

Assessment of genotyping markers in the molecular characterization of a population of clinical isolates of Fusarium in Colombia. / Evaluación de marcadores de genotipificación en la caracterización molecular de una población de aislamientos clínicos de Fusarium en Colombia

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.

Assessment of genotyping markers in the molecular characterization of a population of clinical isolates of Fusarium in Colombia / Evaluación de marcadores de genotipificación en la caracterización molecular de una población de aislamientos clínicos de Fusarium en Colombia

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 sequencing-based reference together with its higher resolution demonstrates its adequacy for the characterization of Fusarium populations.

Introducción. Fusarium es un grupo heterogéneo de hongos, difícil de clasificar y con una amplia gama de estilos de vida, que actúa como saprófito, parásito de plantas o patógeno de humanos y animales. La prevalencia de la fusariosis clínica y la falta de tratamientos han incrementado el interés en su diagnóstico preciso, lo que conlleva la caracterización molecular de las poblaciones. Objetivo. Comparar marcadores de genotipificación en la evaluación de la variabilidad genética e identificación de aislamientos clínicos de Fusarium. Materiales y métodos. Se evaluó la huella genética producida por dos cebadores aleatorios: M13, que amplifica una secuencia minisatélite, y (GACA)4, que corresponde a una secuencia repetitiva de ADN. Utilizando el índice discriminatorio de Hunter Gaston (HGDI), el análisis de varianza molecular (AMOVA) y una prueba de Mantel, se comparó la resolución de estos marcadores con métodos de genotipificación basados en secuenciación y PCR. Resultados. El mayor HGDI se asoció con el marcador M13, seguido de (GACA)4. Las pruebas AMOVA y Mantel mostraron correlación entre las clasificaciones obtenidas con M13 y la referencia basada en la secuenciación parcial del factor de elongación de transcripción 1-alfa (TEF1-α) y el ADNr 28S. Conclusión. La fuerte correlación entre la clasificación obtenida con M13 y el método de referencia, así como su alta resolución, demuestran su idoneidad para la caracterización de poblaciones de Fusarium.

Frequency of Invasive Fungal Disease in Adults: Experience of a Specialized Laboratory in Medellín, Colombia (2009-2015).

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.

Concordance analysis between different methodologies used for identification of oral isolates of Candida species.

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.

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.

Concordance analysis between different methodologies used for identification of oral isolates of Candida species / Análisis de concordancia de diferentes metodologías para la identificación de aislamientos orales de especies de 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.

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.

Epidemiología de la tinea capitis: 19 años de experiencia en un laboratorio clínico especializado en Colombia / Epidemiology of tinea capitis: A 19 years' experience in a Colombian, specialized clinical laboratory

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.

Introduction: There are few written reports on the epidemiology of tinea capitis in Colombia. Objective: To undertake a retrospective study (1994-2013) aimed at describing the behavior of this mycosis and its etiological agents, using a series of patients referred to a specialized diagnostic center in Medellin, Colombia. Methods: This is a retrospective study in which the records were analysed of patients from 1994-2013, who were referred for mycological studies (direct examination and culture) to the Medical and Experimental Mycology Unit of the Corporación para Investigaciones Biológicas (CIB) with the clinical suspicion of tinea capitis. Results: In this period, 415 patients with clinical suspicion of tinea capitis were reported, of which 133 cases were confirmed by the laboratory (32%); most patients 124 (93%) were children, mostly boys 89 (67%). In terms of associated risk factors there was information from 52 confirmed cases, of which 39 (75%) had contact with animals. Direct examination was positive in 87% and fungal culture in 92% of confirmed cases; the etiologic agent most isolated was Microsporum canis (86%), followed by Microsporum gypseum (4%), Trichophyton tonsurans (3%), Trichophyton mentagrophytes (3%), Microsporum audouinii (3%) and Microsporum spp. (1%). Conclusion: Our results represent an important casuistry for the epidemiology of tinea capitis in Colombia. In the absence of more extensive studies on geographic coverage and population characteristics that reveal the true incidence of this mycosis in our country, these data should be considered a valuable contribution to the understanding of the most frequent etiologic agents of tinea capitis in Colombia.

Estandarización y validación en Colombia de una metodología basada en HPLC para la determinación de la concentración sérica de posaconazol / Standardisation and validation of an HPLC method for determining serum posaconazole levels in Colombia

Antecedentes. Hasta la fecha, Colombia no cuenta con un servicio especializado de medición de concentraciones séricas de antifúngicos, procedimiento esencial para el adecuado manejo del tratamiento de las infecciones invasivas por hongos. Objetivos. Estandarizar y validar un protocolo simple, sensible y específico, basado en la cromotografía líquida de alta eficiencia, que, cumpliendo con los parámetros recomendados por la Food and Drug Administration, permita detectar y cuantificar concentraciones séricas de posaconazol. Métodos. Se usó un equipo de cromotografía líquida de alta eficiencia Agilent, serie 1200, con detector ultravioleta de matriz de diodos y columna analítica Eclipse-XDB-C18. Como estándar primario se utilizó posaconazol SCH56592 (lote IRQ-PAZ-10-X-103), y como control interno, itraconazol (lote ZR051211PUC921). La validación se hizo teniendo en cuenta los parámetros recomendados por la Food and Drug Administration (selectividad, curvas de calibración, recuperación, exactitud, precisión, sensibilidad, reproducibilidad y estabilidad de la muestra) para este tipo de métodos. Resultados. Los parámetros cromatográficos más adecuados fueron los siguientes: temperatura de la columna, 25°C; detección ultravioleta, 261nm; volumen de inyección, 50μl; flujo, 0,8ml/min; tiempo de migración, 10min; fase móvil, acetonitrilo:agua (70:30). Los tiempos de retención finales fueron de 3,4 y 7,2min para posaconazol e itraconazol, respectivamente, con un rango de cuantificación amplio y confiable, desde 0,125 hasta 16μg/ml. Bajo estas condiciones el método fue selectivo, el R2 de las curvas de calibración fue≥0,99 y el porcentaje de recuperación fue del 98,7%, con un porcentaje del coeficiente de variación inferior al 10%. El porcentaje de error relativo en la exactitud, así como el porcentaje del coeficiente de variación en la precisión, fueron inferiores al 15%, cumpliendo así con los criterios de aceptación recomendados por la Food and Drug Administration. Conclusiones. La selectividad y pureza de la señal cromatográfica obtenida, así como los límites de detección y cuantificación estandarizados, hacen de esta metodología una excelente herramienta para el seguimiento terapéutico de los pacientes bajo tratamiento con posaconazol (AU)

Background. Colombia currently does not have a specialised service for measuring antifungal levels in serum, which is of prime importance for the proper treatment and correct management of invasive fungal infections. Aims. To standardise and validate a simple, sensitive, and specific protocol, based on high performance liquid chromatography, complying with the parameters recommended by the Food and Drug Administration, to detect, identify, and quantify serum concentrations of posaconazole. Methods. A high performance liquid chromatography Agilent series-1 200 equipment was used with ultraviolet diode array detector and analytical column-Eclipse XDB-C18. Posaconazole-SCH56592 (batch IRQ-PAZ-10-X-103) was used as the primary control and itraconazole (batch ZR051211PUC921) was used as an internal control. The validation was performed taking into account all criteria recommended by the Food and Drug Administration (selectivity, calibration curves, recovery, accuracy, precision, sensitivity, reproducibility, and stability of the sample). Results. The most suitable chromatographic conditions were the following: column temperature 25°C, ultraviolet detection at 261nm, 50μl injection volume, flow volume 0.8ml/min, 10min running time, mobile phase of acetonitrile:water (70:30), and final retention times of 3.4 and 7.2min for posaconazole and itraconazole, respectively, with a wide and reliable quantification range (0.125μg/ml to 16μg/ml). Using these parameters, the method was selective, R2 in the calibration curves was≥0.99, and the percentage recovery was 98.7%, with a coefficient of variation less than 10%. The relative error for accuracy and the coefficient of variation for precision were less than 15%, all meeting the acceptance criteria recommended by the Food and Drug Administration. Conclusions. The selectivity and chromatographic purity of the obtained signal, as well as the standardised limits of detection and quantification, make this method an excellent tool for therapeutic monitoring of patients treated with posaconazole (AU)

[Standardisation and validation of an HPLC method for determining serum posaconazole levels in Colombia]. / Estandarización y validación en Colombia de una metodología basada en HPLC para la determinación de la concentración sérica de posaconazol.

BACKGROUND: Colombia currently does not have a specialised service for measuring antifungal levels in serum, which is of prime importance for the proper treatment and correct management of invasive fungal infections. AIMS: To standardise and validate a simple, sensitive, and specific protocol, based on high performance liquid chromatography, complying with the parameters recommended by the Food and Drug Administration, to detect, identify, and quantify serum concentrations of posaconazole. METHODS: A high performance liquid chromatography Agilent series-1 200 equipment was used with ultraviolet diode array detector and analytical column-Eclipse XDB-C18. Posaconazole-SCH56592 (batch IRQ-PAZ-10-X-103) was used as the primary control and itraconazole (batch ZR051211PUC921) was used as an internal control. The validation was performed taking into account all criteria recommended by the Food and Drug Administration (selectivity, calibration curves, recovery, accuracy, precision, sensitivity, reproducibility, and stability of the sample). RESULTS: The most suitable chromatographic conditions were the following: column temperature 25°C, ultraviolet detection at 261nm, 50µl injection volume, flow volume 0.8ml/min, 10min running time, mobile phase of acetonitrile:water (70:30), and final retention times of 3.4 and 7.2min for posaconazole and itraconazole, respectively, with a wide and reliable quantification range (0.125µg/ml to 16µg/ml). Using these parameters, the method was selective, R2 in the calibration curves was≥0.99, and the percentage recovery was 98.7%, with a coefficient of variation less than 10%. The relative error for accuracy and the coefficient of variation for precision were less than 15%, all meeting the acceptance criteria recommended by the Food and Drug Administration. CONCLUSIONS: The selectivity and chromatographic purity of the obtained signal, as well as the standardised limits of detection and quantification, make this method an excellent tool for therapeutic monitoring of patients treated with posaconazole.

Childhood histoplasmosis in Colombia: Clinical and laboratory observations of 45 patients.

Histoplasmosis is an important mycosis in the Americas; and in children with no immune system abnormalities, histoplasmosis is typically a self-limited process. In contrast, in children with immune problems, disease manifestations are frequently more severe and include dissemination. From 1984 to 2010, a retrospective study of paediatric patients who had been diagnosed with histoplasmosis was performed. A total of 45 pediatric cases of histoplasmosis were identified. The most important risk factor was malnutrition (37%), followed by environmental exposure (33%). The patients exhibited pulmonary infiltrates (83%), fever (76%), cough, constitutional symptoms (38%), headache (35%), and lymph node hypertrophy (33%). Concerning the clinical forms, 64% of the patients presented with the progressive disseminated form that frequently affected the central nervous system (48%). Diagnostic laboratory tests indicated that the cultures were positive for 80% of the patients, the agar gel immunodiffusion was reactive in 95%, the M band of the precipitate was more commonly observed (81%), and the complement fixation tests were reactive in 88% of the patients. The timely diagnosis of histoplasmosis is important, and for this reason, it is hoped that the results of this study will lead pediatricians toward a better understanding of this mycosis in children.

Human neutrophils produce extracellular traps against Paracoccidioides brasiliensis.

Neutrophils play an important role as effector cells and contribute to the resistance of the host against microbial pathogens. Neutrophils are able to produce extracellular traps (NETs) in response to medically important fungi, including Aspergillus spp., Candida albicans and Cryptococcus gattii. However, NET production in response to Paracoccidioides brasiliensis has yet to be studied. We have demonstrated that human neutrophils produce NETs against both conidia and yeasts of P. brasiliensis. Although the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) did not alter NET production against conidia, it partially suppressed NET formation against P. brasiliensis yeasts. Cytochalasin D or IFN-γ did not affect the production of NETs against the fungus. Additionally, a mutant strain of P. brasiliensis with reduced expression of an alternative oxidase induced significantly higher levels of NETs in comparison with the WT strain. Finally, c.f.u. quantification of P. brasiliensis showed no significant differences when neutrophils were treated with DPI, DNase I or cytochalasin D as compared with untreated cells. These data establish that NET formation by human neutrophils appears to be either dependent or independent of reactive oxygen species production, correlating with the fungal morphotype used for stimulation. However, this mechanism was ineffective in killing the fungus.

Detection of antibodies against Paracoccidioides brasiliensis melanin in in vitro and in vivo studies during infection.

Several cell wall constituents, including melanins or melanin-like compounds, have been implicated in the pathogenesis of a wide variety of microbial diseases caused by diverse species of pathogenic bacteria, fungi, and helminthes. Among these microorganisms, the dimorphic fungal pathogen Paracoccidioides brasiliensis produces melanin in its conidial and yeast forms. In the present study, melanin particles from P. brasiliensis were injected into BALB/c mice in order to produce monoclonal antibodies (MAbs). We identified five immunoglobulin G1 (IgG1) κ-chain and four IgM melanin-binding MAbs. The five IgG1 κ-chain isotypes are the first melanin-binding IgG MAbs ever reported. The nine MAbs labeled P. brasiliensis conidia and yeast cells both in vitro and in pulmonary tissues. The MAbs cross-reacted with melanin-like purified particles from other fungi and also with commercial melanins, such as synthetic and Sepia officinalis melanin. Melanization during paracoccidioidomycosis (PCM) was also further supported by the detection of IgG antibodies reactive to melanin from P. brasiliensis conidia and yeast in sera and bronchoalveolar lavage fluids from P. brasiliensis-infected mice, as well as in sera from human patients with PCM. Serum specimens from patients with other mycoses were also tested for melanin-binding antibodies by enzyme-linked immunosorbent assay, and cross-reactivities were detected for melanin particles from different fungal sources. These results suggest that melanin from P. brasiliensis is an immunologically active fungal structure that activates a strong IgG humoral response in humans and mice.

Combined itraconazole-pentoxifylline treatment promptly reduces lung fibrosis induced by chronic pulmonary paracoccidioidomycosis in mice.

Fibrosis is a severe and progressive sequel of many pulmonary diseases, has no effective therapy at present and, consequently, represents a serious health problem. In Latin America, chronic pulmonary paracoccidioidomycosis (PCM) is one of the most important, prevalent and systemic fungal diseases that allows the development of lung fibrosis, with the additional disadvantage that this sequel may appear even after an apparently successful course of antifungal therapy. In this study, was propose the pentoxifylline as complementary treatment in the pulmonary PCM due to its immunomodulatory and anti-fibrotic properties demonstrated in vitro and in vivo in liver, skin and lung. Our objective was to investigate the possible beneficial effects that a combined antifungal (Itraconazole) and immunomodulatory (Pentoxifylline) therapy would have in the development of fibrosis in a model of experimental chronic pulmonary PCM in an attempt to simulate the naturally occurring events in human patients. Two different times post-infection (PI) were chosen for starting therapy, an "early time" (4 weeks PI) when fibrosis was still absent and a "late time" (8 weeks PI) when the fibrotic process had started. Infected mice received the treatments via gavage and were sacrificed during or upon termination of treatment; their lungs were then removed and processed for immunological and histopathologic studies in order to assess severity of fibrosis. When pulmonary paracoccidioidomycosis had evolved and reached an advanced stage of disease before treatment began (as normally occurs in many human patients when first diagnosed), the combined therapy (itraconazole plus pentoxifylline) resulted in a significantly more rapid reduction of granulomatous inflammation and pulmonary fibrosis, when compared with the results of classical antifungal therapy using itraconazole alone.

Detección e identificación de Histoplasma capsulatum por el laboratorio: de los métodos convencionales a las pruebas moleculares / Detection and identification of the fungal pathogen Histoplasma capsulatum: from conventional to molecular methods

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.

Histopathologic and immunologic effects of the itraconazole treatment in a murine model of chronic pulmonary paracoccidioidomycosis.

A comparative study, based on histopathologic findings (inflammation, cellularity, and fibrosis) and immunologic parameters (pro-inflammatory and anti-inflammatory cytokines), was carried out in order to evaluate the effects of itraconazole (ITC) treatment and its starting time in a BALB/c murine model of chronic pulmonary paracoccidioidomycosis (PCM), induced by intranasal inoculation of Paracoccidioides brasiliensis (Pb) conidia. Two different groups of mice were exposed to ITC therapy beginning at the 4th or 8th week after Pb infection, respectively. ITC was administered daily, via gavage, for a period of sixty days. At weeks 0, 4, 8, 12 and 16 the animals were sacrificed and their lungs removed for histology staining with hematoxylin and eosin (H&E), Masson's trichromic and Gomori-Grocott; pulmonary levels of IL-1ß, TNF-α, IFN-γ, IL-13 and TGF-ß were also measured by ELISA. The development or absence of the principal pulmonary PCM sequela, lung fibrosis, was directly related to the therapy's starting time. This and other histopathologic findings were related to the behavior of cytokine levels.

Gene expression analysis of Paracoccidioides brasiliensis transition from conidium to yeast cell.

Paracoccidioides brasiliensis infectious process relies on the initial expression of virulence factors that are assumed to be controlled by molecular mechanisms through which the conidia and/or mycelial fragments convert to yeast cells. In order to analyze the profile of the thermally-induced dimorphic gene expression, 48 h C-L transition cultures which had been incubated at 36 degrees C were studied. By this time approximately 50% of the conidial population had already reverted to yeast form cells. At this transition time, an EST-Orestes library was constructed and characterized. As a result, 79 sequences were obtained, of which 39 (49.4%) had not been described previously in other libraries of this fungus and which could represent novel exclusive C-Y transition genes. Two of these sequences are, among others, cholestanol delta-isomerase, and electron transfer flavoprotein-ubiquinoneoxidoreductase (ETF-QO). The other 40/79 (50.6%) sequences were shared with Mycelia (M), Yeast (Y) or Mycelia to yest transition (M-Y) libraries. An important component of this group of sequences is a putative response regulator receiver SKN7, a protein of high importance in stress adaptation and a regulator of virulence in some bacteria and fungi. This is the first report identifying genes expressed during the C-Y transition process, the initial step required to understand the natural history of P. brasiliensis conidia induced infection.

Co-existence of integumentary lesions and lung x-ray abnormalities in patients with paracoccidioidomycosis (PCM).

In paracoccidioidomycosis (PCM), the primary lung infection remains silent. In this study, attempts were done to define the primary target organ by correlating lung radiographic abnormalities with the time course of mucosal/skin lesions concurrently exhibited at diagnosis by 63 patients in whom microscopy and/or isolation of Paracoccidioides brasiliensis from respiratory secretions had been positive. Mucosal and skin lesions were found in 65.1% and 12.7% of the patients, respectively. Odynophagia and dysphagia were present in 38.1% each. All patients had lung interstitial infiltrates, and 31.7% had also alveolar lesions; fibrosis was recorded in 46% of them. An inverse correlation was shown for fibrosis and presence of either odynophagia or dysphagia. Cluster analyzes strongly supported two sets of patients: those with mucosal damage, odynophagia/dysphagia, and alveolo-interstitial infiltrates and those with dermal lesions, dyspnea, and lung fibrosis. These groups may represent novel stages in the natural course of PCM.

Involvement of extracellular matrix proteins in the course of experimental paracoccidioidomycosis.

We aimed at determining involvement of extracellular matrix proteins (ECMp) and an ECM-binding adhesin (32-kDa protein) from Paracoccidioides brasiliensis, in the course of experimental paracoccidioidomycosis. BALB/c mice were infected with P. brasiliensis conidia previously incubated with soluble laminin, fibronectin and fibrinogen or a mAb against the fungal adhesin. Inflammatory response, chitin levels and cytokine production at different postinfection periods were determined. Chitin was significantly decreased in lungs of mice infected with ECMp-treated conidia when compared with controls at week 8, especially with laminin and fibrinogen. Contrariwise, when animals were infected with mAb-treated conidia no differences in chitin content were found. The observed inflammatory reaction in lungs was equivalent in all cases. IFN-gamma increased significantly in lungs from mice infected with soluble ECMp - (at day 4 and week 12) or mAb-treated conidia (at week 12) when compared with animals infected with untreated conidia. Significant increased levels of tumour necrosis factor-alpha were observed at 8 weeks in animals infected with ECMp-treated conidia while no differences were observed during the remaining periods. These findings point toward an inhibitory effect of ECMp on P. brasiliensis conidia infectivity and suggest that these proteins may interfere with conidia initial adhesion to host tissues probably modulating the immune response in paracoccidioidomycosis.

Role of iron in the nitric oxide-mediated fungicidal mechanism of IFN-gamma-activated murine macrophages against Paracoccidioides brasiliensis conidia.

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 50 U/mL of IFN-gamma or treated with 35 microM 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 FeS04. 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.

Role of iron in the nitric oxide-mediated fungicidal mechanism of IFN-gamma-activated murine macrophages against Paracoccidioides brasiliensis conidia

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 æM 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 percent, respectively) in comparison with non-activated or untreated Mthetas (80 percent). 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 æM 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 por cento, respectivamente) em comparação com macrófagos controle (80 por cento). 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.