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.

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.

Detection of Histoplasma capsulatum in Organic Fertilizers by Hc100 Nested Polymerase Chain Reaction and Its Correlation with the Physicochemical and Microbiological Characteristics of the Samples.

Histoplasma capsulatum is the causative agent of histoplasmosis and this fungus inhabits soils rich in phosphorus and nitrogen that are enriched with bird and bat manure. The replacement of organic matter in agroecosystems is necessary in the tropics, and the use of organic fertilizers has increased. Cases and outbreaks due to the presence of the fungus in these components have been reported. The Instituto Colombiano Agropecuario resolution 150 of 2003 contains the parameters set by the Colombian Technical Standard (NTC 5167) on the physicochemical and microbiological features of fertilizers, but it does not regulate the search for H. capsulatum. The aim of this study was to demonstrate H. capsulatum presence in organic fertilizers by nested polymerase chain reaction (PCR). A total of 239 samples were collected: 201 (84.1%) corresponded to organic fertilizers, 30 (12.5%) to bird excrement, and 8 (3.4%) to cave soils. The Hc100 nested PCR had a detection limit of 0.1 pg/µL and a specificity of 100%. A total of 25 (10.5%) samples were positive and validated by sequencing. Seven of the positive samples represented locations where H. capsulatum was previously detected, suggesting the persistence of the fungus. No significant correlations were detected between the physicochemical and microbiological parameters with the presence of H. capsulatum by nested PCR, indicating the fungus existence in organic fertilizers that complied with the NTC 5167. The Hc100 nested PCR targeting H. capsulatum standardized in this work will improve the evaluation of organic fertilizers and ensure the prevention of outbreaks and cases due to manufacturing, marketing, and use of fertilizers contaminated with H. capsulatum.

Comparative study of Candida spp. isolates: Identification and echinocandin susceptibility in isolates obtained from blood cultures in 15 hospitals in Medellín, Colombia.

OBJECTIVES: Invasive candidiasis has a high impact on morbidity and mortality in hospitalised patients. Accurate and timely methods for identification of Candida spp. and determination of echinocandin susceptibility have become a priority for clinical microbiology laboratories. METHODS: This study was performed to compare matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) identification with sequencing of the D1/D2 region of the rRNA gene complex 28 subunit in 147 Candida spp. isolates obtained from patients with candidaemia. Antimicrobial susceptibility testing was performed by broth microdilution (BMD) and Etest. Sequencing of the FKS1 and FKS2 genes was performed. RESULTS: The most common species isolated were Candida albicans (40.8%), followed by Candida parapsilosis (23.1%) and Candida tropicalis (17.0%). Overall agreement between the results of identification by MALDI-TOF/MS and molecular identification was 99.3%. Anidulafungin and caspofungin susceptibility by the BMD method was 98.0% and 88.4%, respectively. Susceptibility to anidulafungin and caspofungin by Etest was 93.9% and 98.6%, respectively. Categorical agreement between Etest and BMD was 91.8% for anidulafungin and 89.8% for caspofungin, with lower agreements in C. parapsilosis for anidulafungin (76.5%) and C. glabrata for caspofungin (40.0%). No mutations related to resistance were found in the FKS genes, although 54 isolates presented synonymous polymorphisms in the hotspots sequenced. CONCLUSIONS: MALDI-TOF/MS is a good alternative for routine identification of Candida spp. isolates. DNA sequencing of the FKS genes suggested that the isolates analysed were susceptible to echinocandins; alternatively, unknown resistance mechanisms or limitations related to antifungal susceptibility tests may explain the resistance found in a few isolates.

Genotyping of Fusarium Isolates from Onychomycoses in Colombia: Detection of Two New Species Within the Fusarium solani Species Complex and In Vitro Antifungal Susceptibility Testing.

Fusariosis have been increasing in Colombia in recent years, but its epidemiology is poorly known. We have morphologically and molecularly characterized 89 isolates of Fusarium obtained between 2010 and 2012 in the cities of Bogotá and Medellín. Using a multi-locus sequence analysis of rDNA internal transcribed spacer, a fragment of the translation elongation factor 1-alpha (Tef-1α) and of the RNA-dependent polymerase subunit II (Rpb2) genes, we identified the phylogenetic species and circulating haplotypes. Since most of the isolates studied were from onychomycoses (nearly 90 %), we carried out an epidemiological study to determine the risk factors associated with such infections. Five phylogenetic species of the Fusarium solani species complex (FSSC), i.e., F. falciforme, F. keratoplasticum, F. lichenicola, F. petroliphilum, and FSSC 6 as well as two of the Fusarium oxysporum species complex (FOSC), i.e., FOSC 3 and FOSC 4, were identified. The most prevalent species were FOSC 3 (38.2%) followed by F. keratoplasticum (33.7%). In addition, our isolates were distributed into 23 haplotypes (14 into FOSC and nine into FSSC). Two of the FSSC phylogenetic species and two haplotypes of FSSC were not described before. Our results demonstrate that recipients of pedicure treatments have a lower probability of acquiring onychomycosis than those not receiving such treatments. The antifungal susceptibility of all the isolates to five clinically available agents showed that amphotericin B was the most active drug, while the azoles exhibited lower in vitro activity.

Whole-Genome Sequencing to Determine Origin of Multinational Outbreak of Sarocladium kiliense Bloodstream Infections.

We used whole-genome sequence typing (WGST) to investigate an outbreak of Sarocladium kiliense bloodstream infections (BSI) associated with receipt of contaminated antinausea medication among oncology patients in Colombia and Chile during 2013-2014. Twenty-five outbreak isolates (18 from patients and 7 from medication vials) and 11 control isolates unrelated to this outbreak were subjected to WGST to elucidate a source of infection. All outbreak isolates were nearly indistinguishable (<5 single-nucleotide polymorphisms), and >21,000 single-nucleotide polymorphisms were identified from unrelated control isolates, suggesting a point source for this outbreak. S. kiliense has been previously implicated in healthcare-related infections; however, the lack of available typing methods has precluded the ability to substantiate point sources. WGST for outbreak investigation caused by eukaryotic pathogens without reference genomes or existing genotyping methods enables accurate source identification to guide implementation of appropriate control and prevention measures.

Implementation of a Training Course Increased the Diagnosis of Histoplasmosis in Colombia.

Histoplasmosis causes a significant mortality, especially persons living with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) from developing countries where access to both appropriate diagnostic methods and antiretroviral therapy are limited. A total of 81 physicians assigned to 17 Colombian departments (states) received training in the clinical, epidemiological, and diagnostic aspects of histoplasmosis. Once this training was received and during the period of October 2009-November 2012, these physicians sent biological samples for immunodiagnostic, mycological, and molecular tests from their patients with suspicion of histoplasmosis. A total of 1,536 samples from 768 patients were evaluated. Of the 768 patients studied, 463 (60%) were HIV positive, 214 (28%) HIV negative, and in 91 (12%) this diagnosis was unknown, and 538 (70%) were males. The 1,536 specimens studied comprised 722 sera, 439 blood samples, and 241 urines, which were tested by immunodiffusion (ID), culture, and antigenuria, respectively; in addition, 134 specimens were tested by performing a molecular assay. Histoplasmosis was diagnosed in 133 patients (17%). After the training, we observed more diagnoses from 27 to 44 cases per year. In this study, a significantly increased number of histoplasmosis cases reported by year were observed after implementing an educational training program.

Association of clinical and demographic factors in invasive candidiasis caused by fluconazole-resistant Candida species: a study in 15 hospitals, Medellín, Colombia 2010-2011.

Candida is the most important agent of fungal infections. Several risk factors have been described associated with invasive infection by fluconazole-resistant Candida spp. A prospective cross-sectional study with case-control analysis was conducted. Case group patients with fluconazole-resistant Candida isolate were included; control group were patients with fluconazole-susceptible Candida spp. A multivariate logistic regression model was performed. Three hundred isolates of Candida spp. were analyzed. Most frequent species were Candida albicans/Candida dubliniensis (48.3%) and Candida tropicalis (22.3%). Posaconazole susceptibility was 93.7%; voriconazole, 84%; and fluconazole, 78.7%. Susceptibility to anidulafungin and caspofungin was 92.7% and 92.3%, respectively. Neutropenia (adjusted odds ratio [aOR] 6.5, 95% confidence interval [CI] 1.0-43.1), antifungal exposure (aOR 5.1, 95% CI 2.3-11.2), and antituberculosis therapy (aOR 7.7, 95% CI 1.4-43.2) were associated to fluconazole resistance. Susceptibility results are useful to guide the selection of empiric antifungal treatment and the design of local therapeutic guidelines. Previous antifungal exposure suggests possible resistance to fluconazole, pointing towards the selection of a different class of antifungal agents.

Diagnostic value of culture and serological tests in the diagnosis of histoplasmosis in HIV and non-HIV Colombian patients.

We determined the value of culture and serological tests used to diagnose histoplasmosis. The medical records of 391 histoplasmosis patients were analyzed. Diagnosis of the mycosis was assessed by culture, complement fixation, and immunodiffusion tests; 310 patients (79.5%) were male, and 184 patients (47.1%) were infected with human immunodeficiency virus (HIV). Positivity value for cultures was 35.7% (74/207), reactivity of serological tests was 95.2% (160/168), and a combination of both methodologies was 16.9% (35/207) for non-HIV patients. Positivity value for cultures was 75.0% (138/184), reactivity of serological tests was 92.4% (85/92), and a combination of both methodologies was 26.0% (48/184) for HIV/acquired immunodeficiency syndrome (AIDS) patients; 48.1% (102/212) of extrapulmonary samples from HIV/AIDS patients yielded positive cultures compared with 23.1% (49/212) in non-HIV patients. Lymphocyte counts made for 33.1% (61/184) of HIV/AIDS patients showed a trend to low CD4+ numbers and higher proportion of positive cultures. These results indicate that culture is the most reliable fungal diagnostic method for HIV/AIDS patients, and contrary to what is generally believed, serological assays are useful for diagnosing histoplasmosis in these patients.

Cryptococcosis in Colombia: Results of the national surveillance program for the years 2006-2010 / Criptococosis en Colombia: resultados de la encuesta nacional, 2006-2010

Introduction: A survey on cryptococcosis is being conducted regularly in Colombia since 1997. We present hereby the results corresponding to patients diagnosed from 2006 to 2010. Objective: To analyze the data obtained during this period. Materials and methods: Retrospective analysis of the corresponding surveys. Results: A total of 526 surveys originating from 72% of the Colombian political divisions were received during the 5-year period. Most patients (76.6%) were males and 74.9% were 21-50 years old. The most prevalent risk factor was HIV infection (83.5%) with cryptococcosis defining AIDS in 23% of the cases. In the general population the estimated mean annual incidence rate for cryptococcosis was 2.4 x 106 inhabitants while in AIDS patients this rate rose to 3.3 x 103. In 474 surveys stating clinical features, most frequent complaints were headache 84.5%, fever 63.4%, nausea and vomiting 57.5%, mental alterations 46.3%, meningeal signs 33.0%, cough 26.4% and visual alterations 24.5%. Neurocryptococcosis was recorded in 81.8% of the cases. Laboratory diagnosis was based on direct examination, culture and latex in 29.3% cases. From 413 Cryptococcus isolates analyzed, 95.6% were identified as C. neoformans var. grubii, 1% C. neoformans var. neoformans, and 3.4% C. gattii. Treatment was reported for 71.6% of the cases with amphotericin B alone or in combination with fluconazole prescribed in 28%. Conclusions: Surveys done through passive surveillance continue to be sentinel markers for HIV infection and represent a systematic approach to the study of opportunistic problems regularly afflicting AIDS patients since cryptococcosis requires no compulsory notification in Colombia.

Introducción. Desde 1997 se viene realizando un programa nacional de vigilancia sobre la criptococosis en Colombia. Se presentan los resultados correspondientes a los pacientes diagnosticados entre el 2006 y el 2010. Objetivo. Analizar los datos obtenidos durante este periodo. Materiales y métodos. Análisis retrospectivo de las encuestas. Resultados. Durante los cinco años mencionados se recibieron 526 encuestas representativas del 72 % de la división política colombiana. La mayoría de pacientes (76,6 %) eran hombres y 74,9 % estaban entre los 21 y los 50 años. El factor de riesgo prevalente fue la infección por VIH (83,5 %), y la criptococosis definió el sida en 23 % de los casos. La incidencia anual promedio en la población general fue de 2,4 por un millón de habitantes mientras que, en pacientes con sida, aumentó a 3,3 por 1.000. En 474 encuestas se informaron manifestaciones clínicas; las más frecuentes fueron: cefalea (84,5 %), fiebre (63,4 %), náuseas y vómito (57,5 %), alteraciones mentales (46,3 %), signos meníngeos (33 %), tos (26,4 %) y alteraciones visuales (24,5 %). La neurocriptococosis se reportó en 81,8 % de los casos. El diagnóstico se hizo por examen directo, cultivo y antigenemia en 29,3 % de los casos. De 413 aislamientos recuperados, 95,6 % fueron C. neoformans var. grubii, 1 % C. neoformans var. neoformans, y 3,4 % C. gattii. En 71,6 % de los casos para el tratamiento se administró anfotericina B y en 28 % se combinó con fluconazol. Conclusiones. La vigilancia pasiva continúa siendo un marcador centinela para la infección por VIH, y constituye una aproximación sistemática al estudio de infecciones oportunistas en pacientes con sida, debido a que la criptococosis no es de notificación obligatoria en Colombia.

Cryptococcosis in Colombia: results of the national surveillance program for the years 2006-2010.

INTRODUCTION: A survey on cryptococcosis is being conducted regularly in Colombia since 1997. We present hereby the results corresponding to patients diagnosed from 2006 to 2010. OBJECTIVE: To analyze the data obtained during this period. MATERIALS AND METHODS: Retrospective analysis of the corresponding surveys. RESULTS: A total of 526 surveys originating from 72% of the Colombian political divisions were received during the 5-year period. Most patients (76.6%) were males and 74.9% were 21-50 years old. The most prevalent risk factor was HIV infection (83.5%) with cryptococcosis defining AIDS in 23% of the cases. In the general population the estimated mean annual incidence rate for cryptococcosis was 2.4 x 106 inhabitants while in AIDS patients this rate rose to 3.3 x 103. In 474 surveys stating clinical features, most frequent complaints were headache 84.5%, fever 63.4%, nausea and vomiting 57.5%, mental alterations 46.3%, meningeal signs 33.0%, cough 26.4% and visual alterations 24.5%. Neurocryptococcosis was recorded in 81.8% of the cases. Laboratory diagnosis was based on direct examination, culture and latex in 29.3% cases. From 413 Cryptococcus isolates analyzed, 95.6% were identified as C. neoformans var. grubii, 1% C. neoformans var. neoformans, and 3.4% C. gattii. Treatment was reported for 71.6% of the cases with amphotericin B alone or in combination with fluconazole prescribed in 28%. CONCLUSIONS: Surveys done through passive surveillance continue to be sentinel markers for HIV infection and represent a systematic approach to the study of opportunistic problems regularly afflicting AIDS patients since cryptococcosis requires no compulsory notification in Colombia.

Histoplasmosis en Colombia: resultados de la encuesta nacional, 1992-2008 / Histoplasmosis: results of the Colombian National Survey, 1992-2008

Introducción. La histoplasmosis está caracterizada por variadas manifestaciones que van desde la afección subclínica a la enfermedad diseminada, y suele presentarse tanto en huéspedes inmunocompetentes como inmunosuprimidos. Como la enfermedad no es de notificación obligatoria en Colombia, se diseñó una encuesta para recolectar información de los casos diagnosticados en el país. Objetivo. El objetivo de este trabajo fue analizar los datos recolectados desde 1992 hasta 2008. Materiales y métodos. La encuesta incluyó datos demográficos, factores de riesgo, manifestaciones clínicas, estudios de imágenes, métodos diagnósticos y tratamiento antifúngico. Los pacientes se agruparon de acuerdo con los factores de riesgo y se compararon los correspondientes hallazgos.Resultados. Se examinaron 434 encuestas provenientes de 20 de los departamentos colombianos. La mayoría (96,1 %) correspondían a adultos, 77 % eran hombres con edad promedio de 38,4 años, y sólo 3,9 % eran niños o adolescentes. En los adultos, 70,5 % tenían sida y 7 % presentaban otra inmunosupresión. Las manifestaciones predominantes fueron fiebre (76,1 %), tos (54,8 %) y síntomas constitucionales (56,8 %). En las radiografías, las anormalidades fueron principalmente infiltrados (65,9 %) y nódulos (17,1 %). El diagnóstico se estableció por observación microscópica de H. capsulatum en 49,6 % casos, por cultivo en 58 % y por pruebas serológicas en 14,6 %. El tratamiento se informó sólo en 52,5 % casos. Conclusiones. Se demostró que la histoplasmosis es frecuente en Colombia, especialmente en grupos de riesgo como son los pacientes infectados con el VIH. El análisis de este número relevante de pacientes permitió establecer comparaciones válidas sobre aspectos de la histoplasmosis en nuestro país.

Introduction. Histoplasmosis, a fungal disorder characterized by a wide spectrum of manifestations that range from subclinical infections to disseminated processes, affects both immunocompetent and immunosuppressed individuals. Histoplasmosis is not a reportable disease in Colombia and consequently, a survey was designed to collect histoplasmosis cases diagnosed in the country. Objective. The aim of this work was to analyze the data collected from 1992 to 2008. Materials and methods. The survey included demographic data, risk factors, clinical manifestations, imaging data, diagnostic methods and antifungal treatment. Patients were grouped according to risk factors and comparisons of the various findings were done. Results. A total of 434 surveys were gathered from 20 of the country’s Departments. Most patients (96.1%) were adults, 77% were males with a mean age of 38.4 years. Only 3.9% were children less than 15 years of age. In the adult population, AIDS was reported in 70.5% of the cases; additionally, in 7.0% patients other immunosuppressive conditions were informed. The most frequent clinical manifestations were fever (76.1%), cough (54.8%) and constitutional symptoms (56.8%). X rays abnormalities were represented mainly by infiltrates (65.9%) and nodules (17.1%). Diagnosis was made by microscopic observation of H. capsulatum in 49.6% patients, by culture in 58.0% and by serological test in 14.6% cases. Antifungal use was recorded in 52.5% cases. Conclusions. Histoplasmosis is frequent in Colombia, especially in certain risk factor groups such as the HIV-infected population. Data collected from this large number of cases has allowed valid comparisons on various aspects of histoplasmosis in Colombia.

Histoplasmosis diseminada progresiva en una cohorte de pacientes coinfectados con el VIH / Progressive disseminated histoplasmosis in a cohort of patients with HIV coinfection

Objetivo: identificar manifestaciones clínicas y métodos de laboratorio conducentes al diagnóstico de histoplasmosis diseminada progresiva (HDP), en una cohorte de pacientes coinfectados con el VIH. Diseño: análisis retrospectivo de historias clínicas. No intervención adicional. Pacientes: cuarenta pacientes con HDP e infección por VIH del Hospital La María, enero de 1992 a diciembre de 2008. Mediciones: datos demográficos, signos, síntomas y exámenes de laboratorio que permitieron el diagnóstico de HDP. Resultados: cuarenta pacientes, 34 hombres (85%), y seis mujeres (15%), con edades promedio de 33.4 y 27 años, respectivamente. En éstos predominaron: tos (77.5%), fiebre (90%) y anorexia con pérdida de peso en 92.5% y 77.4%, respectivamente. Lesiones en piel en 55% y en mucosa en 50%, crecimiento ganglionar en 62.5% y hepatomegalia en 52.5%. Menos frecuentes fueron disnea, esplenomegalia, vómito, diarrea y cefalea. Presentaron anemia el 85%, leucopenia el 52.5% y trombocitopenia el 30% de los pacientes. Exámenes micológicos: directo positivo en 21 muestras, de lavado broncoalveolar siete, piel seis, ganglio 12, biopsia transbronquial una y lesión de mucosa una. Se aisló H. capsulatum en todos los pacientes a partir de muestras de piel 10, ganglio 18, sangre tres, médula ósea una, lavado broncoalveolar (LBA) 10, y mucosa tres. Además, en siete pacientes, el hongo se aisló de más de un sitio anatómico. La serología realizada en 13 pacientes, se mostró reactiva en la inmunodifusión en gel de agar (IDGA) en 11 y en la fijación del complemento (FC) en 10. Conclusión: ante un cuadro clínico compatible con HDP en paciente con infección por VIH que presenta fiebre, pérdida del estado general, crecimiento ganglionar, compromiso medular y piel y mucosas, el laboratorio permitirá confirmar fácilmente el diagnóstico de la sospecha clínica de la entidad (Acta Med Colomb 2011; 36: 63-67).

Objective: to identify the clinical manifestations and laboratory methods leading to the diagnosis of progressive disseminated histoplasmosis (PDH) in a cohort of patients co-infected with HIV. Design: retrospective analysis of case histories. No further intervention. Patients: forty patients with PDH and HIV infection from Hospital La María. January 1992 to December 2008. Measurements: demographic data, signs and symptoms, and laboratory tests leading to the diagnosis of PDH. Results: 40 patients, 34 males (85%) and 6 females (15%), with mean ages of 33.4 and 27 years, respectively. The dominant symptoms were: cough (77.5%), fever (90%), and anorexia with weight loss in 92.5% and 77.4%, respectively. Skin lesions in 55% and mucosal lesions in 50%, enlargement of lymph nodes in 62.5%, and hepatomegaly in 52.5%. Less frequent manifestations were dyspnea, splenomegaly, vomiting, diarrhea, and headache. Anemia was found in 85%, leucopenia in 52.5%, and thrombocytopenia in 30% of the patients. Mycological tests: directly positive in 21 samples: bronchoalveolar lavage (7), skin (6), lymph node (12), transbronchial biopsy (1), and mucosal lesion (1). H. capsulatum was isolated in all patients from samples of: skin (10), lymph node (18), blood (3), bone marrow (1), bronchoalveolar lavage fluid (10), and mucosa (3). Moreover, the fungus was isolated in more than one anatomical site in 7 patients. Serology carried out in 13 patients was reactive on agar gel immunodifusion test in 11 cases and on complement fixation in 10 cases. Conclusion: in the face of a clinical picture consistent with PDH in a patient with HIV who presents with fever, constitutional symptoms, enlargement of lymph nodes, and involvement of the bone marrow, skin, and mucous membranes, laboratory testing allows easy confirmation of the clinically suspected condition (Acta Med Colomb 2011; 36: 63-67).

Histoplasmosis: results of the Colombian national survey, 1992-2008.

INTRODUCTION: Histoplasmosis, a fungal disorder characterized by a wide spectrum of manifestations that range from subclinical infections to disseminated processes, affects both immunocompetent and immunosuppressed individuals. Histoplasmosis is not a reportable disease in Colombia and consequently, a survey was designed to collect histoplasmosis cases diagnosed in the country. OBJECTIVE: The aim of this work was to analyze the data collected from 1992 to 2008. Materials and methods. The survey included demographic data, risk factors, clinical manifestations, imaging data, diagnostic methods and antifungal treatment. Patients were grouped according to risk factors and comparisons of the various findings were done. RESULTS: A total of 434 surveys were gathered from 20 of the country's Departments. Most patients (96.1%) were adults, 77% were males with a mean age of 38.4 years. Only 3.9% were children less than 15 years of age. In the adult population, AIDS was reported in 70.5% of the cases; additionally, in 7.0% patients other immunosuppressive conditions were informed. The most frequent clinical manifestations were fever (76.1%), cough (54.8%) and constitutional symptoms (56.8%). X rays abnormalities were represented mainly by infiltrates (65.9%) and nodules (17.1%). Diagnosis was made by microscopic observation of H. capsulatum in 49.6% patients, by culture in 58.0% and by serological test in 14.6% cases. Antifungal use was recorded in 52.5% cases. CONCLUSIONS: Histoplasmosis is frequent in Colombia, especially in certain risk factor groups such as the HIV-infected population. Data collected from this large number of cases has allowed valid comparisons on various aspects of histoplasmosis in Colombia.

Candida y candidiasis invasora: un reto continuo para su diagnóstico temprano / Candida and candidiasis: the challenge continues for an early diagnosis

La candidiasis invasora representa el 75% de las infecciones por hongos en pacientes hospitalizados, con una mortalidad que alcanza cifras hasta del 78%. La frecuencia de estas infecciones varía de acuerdo con el servicio de hospitalización y los factores de riesgo de los pacientes. Paralelamente, se han venido observando cambios en la epidemiología de las especies de Candida, variaciones en su prevalencia y en la resistencia a los antimicóticos según su localización geográfica. Por todo lo anterior, es imperativo establecer un diagnóstico temprano que lleve a la identificación correcta de la especie implicada de manera que se instaure un pronto y adecuado tratamiento antimicótico. El diagnóstico de la candidiasis invasora continúa siendo un reto, en el cual combinar los diferentes métodos diagnósticos, los microbiológicos, los inmunológicos y los nuevos moleculares, aún en desarrollo y validación, es la mejor estrategia para lograr un dictamen oportuno. En esta revisión se describen los métodos disponibles, sus limitaciones y las perspectivas de los que están en etapa de desarrollo y validación. En la última década se cuenta con métodos de referencia para la medición de susceptibilidad in vitro a los antimicóticos, lo cual ha permitido conocer los perfiles de sensibilidad de las diferentes especies de Candida a escala mundial y local.

Invasive candidiasis represents 75% of fungal infections in hospitalized patients, with reported mortalities up to 78%. The frequency of these infections varies according to the hospital services and the risk factors of the patients. In parallel, changes in the epidemiology of the Candida species have been observed, in particular variations in their prevalence and in their resistance to antifungals according to geographic location. For these reasons it is crucial to establish an early diagnosis that identifies the pathogen to the species level in order to allow an appropriate therapeutic decision. The diagnosis of invasive candidiasis continues to be a challenge, where combining the different available methods (microbiologic, immunologic and new molecular approaches) is the best strategy to achieve a prompt and accurate diagnosis. We review the currently available assays for conventional and molecular diagnosis, their limitations, and the perspectives for assays that are now in development and validation. In the last decade, well established reference methods have become available for testing antifungal susceptibility and this has allowed worldwide and regional sensitivity profiles to be established for the different Candida species.

Sensibilidad a fluconazol y voriconazol de aislamientos de Candida spp., obtenidos de mucosa oral de pacientes con sida / Sensitivity to Fluconazol and Voriconazol in Candida spp. isolates from the oral mucosa of HIV positive patients

Se determinó la sensibilidad al fluconazol y al voriconazol de aislamientos de Candida spp. obtenidosde la mucosa oral de 54 pacientes con sida hospitalizadosen la ESE Hospital La María (Medellín, Colombia). además, se comprobó la especie de tales aislamientos.Los pacientes eran todos adultos (promedio de 40,5 años, rango de 23 a 56) y la mayoría (77,8 por ciento) hombres. En 40 (71,1 por ciento) de ellos se obtuvo crecimiento de Candida spp. y en 6 (11,1por ciento) se aisló más de una especie de Candida. La clasificación a especie reveló C. albicans, C. krusei, C. tropicalis, C.parapsilosis y C. glabrata. La determinación de lasensibilidad in vitro se hizo por difusión en agar con las especificaciones del CLSI de Estados Unidos (M44P). El 72,9 por ciento de los aislamientos de Candida spp. fueron sensibles al fluconazol; 6,3 por ciento, sensibles dependientes de la dosis, y 20,8 por ciento, resistentes. Para el voriconazol, 89,6 por ciento fueron sensibles; 8,3 por ciento, sensibles dependientes de la dosis y 2,1 por ciento, resistentes. La resistencia se observó en C. albicans y C. krusei. Estos resultados demuestran la efectividad de ambos antimicóticos y fue mayor la correspondiente al voriconazol. Igualmente se señala la importancia de las pruebas in vitro puesto que en pacientes con VIH/sida pueden encontrarse especies resistentes. Se ratifica la importancia de la clasificación por especiede las levaduras del género Candida.

[Results of nine years of the clinical and epidemiological survey on cryptococcosis in Colombia, 1997-2005]. / Estudio clínico y epidemiológico de la criptococosis en Colombia: resultados de nueve años de la encuesta nacional, 1997-2005.

INTRODUCTION: A national survey on cryptococcosis has been conducted in Colombia since 1997. The survey data recorded over a 9-year period, 1997 to 2005, was summarized. MATERIALS AND METHODS: The format provided by the European Confederation of Medical Mycology was adapted with the correspondent permission. RESULTS: Over the 9 year period, 931 surveys were received from 76 centers. The associated disease syndromes were as follows: 891 (95.7%) were neurocryptococosis cases, 27 (2.9%) pulmonary disease, 5 (0.5%) cutaneous lesions, 2 (0.2%) ganglionar forms, 2 (0.2%) oropharyngeal lesions and one case (0.1%) each from peritonitis, liver lesion, cellulitis and urinary tract infection. Demographic data indicated 82.7% of the subjects were males, and 59.4% were between 20-39 years old; 25 children less than 16 years old were reported. The prevalent risk factor was HIV infection (78.1%). The mean annual incidence rate of cryptococcosis in the general population was 2.4 X 106 inhabitants, but in AIDS patients the rate rose to one in 3 X 103. The most frequent clinical features were headache (85.2%), nausea and vomiting (59.1%), fever (59.0%), mental changes (46.2%), meningeal signs (33.4%), cough (23.6%) and visual alterations or loss of vision (20.9%). Laboratory data showed that direct examination of cerebrospinal fluid (CSF) was positive in 92.8% cases and Cryptococcus was recovered in 90.3% of the cases. Cryptococcal antigen reactivity was 98.9% in CSF and 93.7% in serum samples. From 788 isolates submitted, 95.9% were C. neoformans var. grubii serotype A, 0.3% var. neoformans serotype D, 3.3% C. gattii serotype B and 0.5% C. gattii serotype C. The majority of patients were treated initially with amphotericin B. CONCLUSION: Cryptococcosis incidence has increased dramatically in Colombia with the AIDS pandemic and it can be considered as a sentinel marker for HIV infection.

Estudio clínico y epidemiológico de la criptococosis en Colombia: resultados de nueve años de la encuesta nacional, 1997-2005 / Results of nine years of the clinical and epidemiological survey on cryptococcosis in Colombia, 1997-2005

Introducción. Con el fin de conocer la situación de la criptococosis en Colombia se ha llevado a cabo una encuesta nacional. El presente estudio analiza la información recogida de 1997 a 2005. Materiales y métodos. Se utilizó el formato de encuesta de la Confederación Europea de Micología Médica. Resultados. Se recibieron 931 encuestas de 76 centros. Se identificaron 891 (95,7 por ciento) casos de neurocriptococosis, 27 (2,9 por ciento) de formas pulmonares, cinco (0,5 por ciento) de lesiones cutáneas, dos (0,2 por ciento) de ganglionares, dos (0,2 por ciento) de úlceras orofaríngeas y un caso (0,1 por ciento) de cada una de las siguientes formas clínicas: peritonitis, lesión hepática, celulitis de los miembros inferiores e infección de las vías urinarias. El 82,7 por ciento fueron hombres con predominio de los adultos jóvenes. Sin embargo, se reportaron 25 (2,7 por ciento) niños. La infección por el virus de la inmunodeficiencia humana se encontró en el 78,1 por ciento de los casos. La incidencia promedio anual de criptococosis en la población general fue de 2,4 casos x106, pero en los pacientes con sida se elevó a 3,0 casos x 103. Las manifestaciones clínicas más frecuentes fueron: cefalea (85,2 por ciento), náuseas y vómito (59,1 por ciento), fiebre (59 por ciento), cambios mentales (46,2 por ciento) y signos meníngeos (33,4 por ciento). El examen directo de líquido cefalorraquídeo fue positivo en el 92,8 por ciento de los casos y el hongo se recuperó en el 90,3 por ciento. El antígeno capsular tuvo una positividad del 98,9 por ciento en el líquido cefalorraquídeo y del 93,7 por ciento en el suero. De 788 aislamientos remitidos, 95,9 por ciento fueron Cryptococcus neoformans var. grubii serotipo A, 0,3 por ciento, var. neoformans serotipo D, 3,3 por ciento, C. gattii serotipo B y 0,5 por ciento, C. gattii serotipo C. La mayoría de los pacientes fueron tratados inicialmente con anfotericina B. Conclusión: La criptococosis es una enfermedad cuya incidencia a...

Introduction. A national survey on cryptococcosis has been conducted in Colombia since 1997. The survey data recorded over a 9-year period, 1997 to 2005, was summarized. Materials and methods. The format provided by the European Confederation of Medical Mycology was adapted with the correspondent permission. Results. Over the 9 year period, 931 surveys were received from 76 centers. The associated disease syndromes were as follows: 891 (95.7%) were neurocryptococosis cases, 27 (2.9%) pulmonary disease, 5 (0.5%) cutaneous lesions, 2 (0.2%) ganglionar forms, 2 (0.2%) oropharyngeal lesions and one case (0.1%) each from peritonitis, liver lesion, cellulitis and urinary tract infection. Demographic data indicated 82.7% of the subjects were males, and 59.4% were between 20-39 years old; 25 children less than 16 years old were reported. The prevalent risk factor was HIV infection (78.1%). The mean annual incidence rate of cryptococcosis in the general population was 2.4 X 106 inhabitants, but in AIDS patients the rate rose to one in 3 X 103. The most frequent clinical features were headache (85.2%), nausea and vomiting (59.1%), fever (59.0%), mental changes (46.2%), meningeal signs (33.4%), cough (23.6%) and visual alterations or loss of vision (20.9%). Laboratory data showed that direct examination of cerebrospinal fluid (CSF) was positive in 92.8% cases and Cryptococcus was recovered in 90.3% of the cases. Cryptococcal antigen reactivity was 98.9% in CSF and 93.7% in serum samples. From 788 isolates submitted, 95.9% were C. neoformans var. grubii serotype A, 0.3% var. neoformans serotype D, 3.3% C. gattii serotype B and 0.5% C. gattii serotype C. The majority of patients were treated initially with amphotericin B. Conclusion. Cryptococcosis incidence has increased dramatically in Colombia with the AIDS pandemic and it can be considered as a sentinel marker for HIV infection.

Disseminated histoplasmosis: a comparative study between patients with acquired immunodeficiency syndrome and non-human immunodeficiency virus-infected individuals.

We studied 52 patients with disseminated histoplasmosis, 30 with the acquired immunodeficiency syndrome (AIDS) (cohort 1) and 22 not co-infected with the human immunodeficiency virus (cohort 2). Demographic, clinical, laboratory, mycologic findings, as well as antifungal therapy and highly active antiretroviral (HAART), were analyzed. Skin lesions were significantly higher in cohort 1 than in cohort 2 (P = 0.001). Anemia, leukopenia, and an elevated erythrocyte sedimentation rate were also more pronounced in cohort 1 than in cohort 2 (P < 0.001). Histoplasma capsulatum was isolated more often in cohort 1 than in cohort 2 (P < 0.05) patients, but antibodies to H. capsulatum were detected more frequently in cohort 2 than in cohort 1 (P < 0.05). Itraconazole treatment was less effective in cohort 1 than in cohort 2 (P = 0.012). In cohort 1 patients, HAART improved response to antifungals when compared with individuals not given HAART (P = 0.003), who exhibited higher mortality rates (P = 0.025). Cohort 1 patients who were given dual antifungal and anti-retroviral therapies responded as well as the non-HIV patients in cohort 2, who were treated only with itraconazole. These results indicate the need to promote restoration of the immune system in patients with AIDS and histoplasmosis.