Comparison between Two Molecular Techniques: Nested and Real-Time Polymerase Chain Reaction Targeting 100-kDa Hc Protein for Detection of Histoplasma capsulatum in Environmental Samples.

Histoplasmosis, one of the most frequent endemic mycoses in the Americas, is caused by the inhalation of airborne conidia of Histoplasma capsulatum. Better understanding of the distribution of this fungus in the environment is important for the development of appropriate public health measures to prevent human infections. Previously, we used Hc100 nested polymerase chain reaction (PCR) to identify H. capsulatum DNA in 10% of environmental samples in Colombia. Here, we validate a 100-kDa real-time PCR assay for the detection of this fungus in the environment. Using this method, we identified H. capsulatum DNA in 80% of samples of raw organic materials, such as chicken manure, soil from caves, and bird and bat guano, as well as in 62% of samples of organic fertilizer that underwent the composting process. We demonstrated that 100-KDa real-time PCR is a useful tool for environmental surveillance that can be used to identify the potential reservoirs of H. capsulatum and to prevent outbreaks, especially in people with the higher risk of exposure, such as spelunkers, farmers, poultry manure collectors, and anyone who handle organic fertilizers or bat and bird excreta.

Molecular epidemiology of Colombian Histoplasma capsulatum isolates obtained from human and chicken manure samples.

The thermally dimorphic fungus Histoplasma capsulatum is the causative agent of histoplasmosis, one of the most prevalent endemic mycosis in the Americas. In tropical regions, agro-ecosystems require organic matter replacement, therefore, the use of organic fertilizers has increased disregarding the fact that certain number of such fertilizers might be contaminated with the fungus, and with their handling resulting in human cases and even outbreaks of histoplasmosis. Additionally, in Colombia, chicken manure is the most common raw material used in the production of organic fertilizers. In this work, we reported the isolation of this fungus from chicken manure, and genetically compared with 42 clinical isolates. The genetically compared environmental isolates grouped together with the clinical ones. Our result suggests that chicken manure may be one of H. capsulatum infection sources. Also, the phylogenetic analyses done with other H. capsulatum isolates indicate that the Colombian isolates are widely distributed in the relational tree thus reveling towards the great genetic diversity among the H. capsulatum Colombian isolates.

Draft Genome Sequences of Two Sporothrix schenckii Clinical Isolates Associated with Human Sporotrichosis in Colombia.

Sporothrix schenckii is a thermodimorphic fungal pathogen with a high genetic diversity. In this work, we present the assembly and similarity analysis of the whole-genome sequences of two clinical isolates from Colombia of S. schenckiisensu stricto.

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.

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: 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.

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.

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.

The naked-tailed armadillo Cabassous centralis (Miller 1899): a new host to Paracoccidioides brasiliensis. Molecular identification of the isolate.

The natural habitat of Paracoccidioides brasiliensis remains undefined but the repeated demonstration of infection by this fungus in the nine-banded armadillo Dasypus novemcinctus has opened interesting research avenues. We report here the isolation of this fungus from the spleen of a naked-tailed armadillo Cabassous centralis (Miller 1899) captured in a coffee farm localized in the Colombian endemic area for paracoccidioidomycosis. This particular isolate was identified by its dimorphism and also by comparison of the PbGP43 gene and ribosomal internal transcribed spacer regions (ITS) with recognized P brasiliensis strains. This finding extends the range of naturally acquired infections in mammals of the family Dasypodidae and confirms the existence of this human pathogen in areas where human paracoccidioidomycosis is known to occur.

Posaconazole therapy for severe abdominal candidiasis: a case report.

We report the successful treatment of a fluconazole-resistant intra-abdominal Candida infection (Candida albicans and Candida tropicalis) with posaconazole (SCH56592) in a 68-year-old woman with a recent history of intra-abdominal surgery.

[Necrotizing fasciitis in an immunocompetent patient caused by Apophysomyces elegans]. / Fascitis necrosante por Apophysomyces elegans, moho de la familia Mucoraceae, en paciente inmunocompetente.

A case study is presented of a 7-year-old boy, seriously injured in a car accident, who developed a fatal infection due to Aphophysomyces elegans--a mold of the Mucoracea family. Fungal invasion was initially manifested by a spotted wound in the left lumbar region which developed into a necrotizing fasciitis. Later this progressed to the right lumbar area, including the gluteus and the corresponding flank. Antimycotic treatment proved ineffective, and the child died 8 weeks after the accident. Other cases due to this fungus are reviewed.

Fascitis necrosante por Apophysomyces elegans, moho de la familia mucoraceae, en paciente inmunocompetente / Necrotizing fasciitis in an immunocompetent patient caused by Apophysomyces elegans

Se presenta la historia de un niño de 7 años de edad, politraumatizado por accidente automovilístico, que falleció a consecuencia de infección por Apophysomyces elegans, un hongo de la familia Mucoracea. La invasión fue progresiva, inicialmente se observó una lesión puntiforme en la región lumbar izquierda que progresó a fascitis necrosante y, posteriormente, afectó la zona lumbar, los glúteos y el flanco derecho. El tratamiento antimicótico resultó inefectivo y el paciente falleció 8 semanas después de su accidente. Se presenta, además, una revisión de los casos atribuidos a este hongo

Mucormycosis (zygomycosis) in a heart-kidney transplant recipient: recovery after posaconazole therapy.

We describe the case of a diabetic patient who developed a severe invasive fungal infection due to Rhizopus species postoperatively after a dual heart/kidney transplantation with subsequent intensive immunosuppressive therapy. No improvement was noted with amphotericin B (deoxycholate) therapy, but salvage treatment with the new azole antifungal posaconazole (200 mg orally 4 times daily) resulted in dramatic clinical improvement as early as 1 week after the initiation of therapy that continued through 23 weeks of treatment, with marked clinical, mycological, and radiological improvements and no adverse events.

[Evaluation of Candida species' susceptibility to fluconazole with the disk diffusion method]. / Evaluación de la susceptibilidad de especies de Candida al fluconazol por el método de difusión de disco.

Infections caused by yeasts belonging to the genus Candida have increased dramatically in the last decades, especially in hospital settings. Concomittantly, antimycotic resistance has emerged, as well as the appearance of non-Candida albicans isolates. To standardize in vitro antifungal susceptibility tests, the agar diffusion test was developed using disks impregnated with the antimycotic compound. Electronic recording of the inhibition zone (BIOMIC), furnishes objective values for the minimal inhibitory concentration (MIC). The fluconazole susceptibility patterns were determined for Candida species isolated from 2.139 patients seen in outpatient clinics or in health-care centers in Colombia, Ecuador and Venezuela. Candida albicans was the species most frequently isolated (62%), followed at a distance by Candida parapsilosis (11%), Candida tropicalis (8.5%), Candida glabata (3.5%) and Candida krusei (2.2%). MIC determinations showed that 88.1% of these isolates were susceptible to fluconazole, 5.1% were susceptible-dose-dependant and 6.8% resistant. An important proportion (92.1%) of the C. albicans isolates proved susceptible while resistance predominated in the remaining species. These results indicate that the BIOMIC method is rapid and simple, constituting a suitable tool for the epidemiologic surveillance of resistance in Candida species.

Evaluación de la susceptibilidad de especies de Candida al fluconazol por el método de difusión de disco / Resistance to fluconazol by Candida species detected using the disk diffusion method

Las infecciones causadas por levaduras del género Candida han aumentado en forma dramática en las últimas décadas, especialmente a nivel hospitalario. Paralelamente con este aumento, se ha notado la aparición de resistencia a los antimicóticos, así como la selección de especies diferentes a Candida albicans, todo lo cual ha hecho necesaria la estandarización de pruebas de susceptibilidad in vitro. Entre las varias pruebas disponibles está la de difusión en agar con discos impregnados de antifúngicos, cuya lectura se hace electrónicamente (BIOMIC), lo que proporciona indicaciones objetivas de la concentración inhibitoria mínima (CIM). Se realizó el estudio para determinar la sensibilidad in vitro al fluconazol de 2.139 aislamientos de especies de Candida provenientes de pacientes atendidos en consulta externa o en salas de hospitalización de centros asistenciales en Colombia, Ecuador y Venezuela (región CELA). Candida albicans fue el microorganismo más frecuentemente aislado (62 por ciento), seguido a distancia por Candida parapsilosis (11 por ciento) , Candida tropicalis (8,5 por ciento), Candida glabrata (3,5 por ciento) y Candida krusei (2,2 por ciento). La CIM demostró que el 88,1 por ciento de estos aislamientos eran susceptibles al fluconazol, 5,1 por ciento eran susceptibles-dosisdependiente y 6,8 por ciento resistentes. El 92,1 por ciento de la especie C. albicans fue susceptible mientras que la resistencia al fluconazol fue más notoria en las restantes especies. El BIOMIC es un método r pido y sencillo para la vigilancia epidemiológica de resistencia en levaduras del género Candida ecuperadas de pacientes atendidos en centros hospitalarios.

[Outbreak of acute histoplasmosis in a family group: identification of the infection source]. / Brote de histoplasmosis aguda en un grupo familiar: identificación de la fuente de infección.

An outbreak of acute histoplasmosis occurring in 4 members of the same family, two women, a girl and a male, is reported. The index case presented acute respiratory symptoms, severe enough to require hospitalization. In the remaining persons, the infection was asymptomatic but was evidenced by reactive histoplasmin serologic tests. Search for the common source of infection led to an enriched soil obtained in a local nursery for growing in-door plants. BALB/c mice were inoculated with suspensions of soils from the potted plants. Histoplasma capsulatum var. capsulatum was isolated from various internal organs of the mice. Although histoplasmosis is observed more frequently in persons with occupations implying risk of exposure and is connected to rural areas, outbreaks and intra-family cases are now common in urban areas. This is due to massive urbanization, deforestation, demolitions and the use of soils enriched with organic compounds, mainly bird/bat excrements. This report calls the attention on the danger involved in using such enriched soils for plant nutrition.

Histoplasmosis: Utilidad de las pruebas serologicas en el diagnostico diferencial / Histoplasmosis: usefull of serological probes in differential diagnosis

La infección por Histoplasma capsulatum se adquiere por inhalación de las estructuras micóticas presentes en el suelo. Su concentración unida a la fuerza de los aerosoles y al estado inmune del hospedero, determinan la intensidad del proceso. La histoplasmosis aguda es más frecuente en agricultores y exploradores, y es propia de áreas rurales. No obstante, las epidemias y los brotes de histoplasmosis suelen también ocurrir en áreas urbanas. La histoplasmosis aguda no siempre es diagnosticada prontamente, lo que permite su diseminación extrapulmonar. Se informan aquí dos casos de histoplasmosis aguda, el primero restringido al pulmón y de pocas semanas de evolución, y el segundo, un proceso de varios meses con diseminación a ganglios linfáticos. Ambos casos fueron diagnosticados sólo después de intervención quirúrgica dado el severo compromiso de los pacientes y la premura para iniciar un tratamiento. No obstante, el diagnóstico diferencial pudiera haber sido abordado de manera más conservadora con base en pruebas de laboratorio.

Brote de histoplasmosis aguda en un grupo familiar: identificación de la fuente de infección / Acute histoplasmosis outbreack in a family group: identification of infection source

Se informa un brote de histoplasmosis ocurrido en los integrantes de una familia y que comprometió a cuatro personas, dos mujeres, una niña y un hombre. El caso índice consultó por sintomatología respiratorio grave, de comienzo súbito, que requirió hospitalización. En los otros casos, la infección curso de manera asintomática pero se puso en evidencia por la reactividad en las pruebas serológicas con histoplasmina. La búsqueda de una fuente común de contagio llevó a sospechar que era la tierra de un vivero que se había utilizado como fertilizante de las plantas caseras. Las supensiones de las tierras de las mecetas sirvieron para inocular ratones BALB/c, de cuyos órganos fue posible aislar el agente etiológico, Histoplasma capsulatum var. capsulatum. Si bien la histoplasmosis es más frecuente en ciertas ocupaciones y es propia de áreas rurales, las epidemias y los brotes son ahora comunes en áreas urbanas debido a actividades como la urbanización masiva, la tala de árboles, las demoliciones y el uso de tierras enriquecidas con abonos orgánicos (gallinaza, guano). Se llama la atención sobre el peligro que representa esta última actividad

El caso de infecciosas: esporotricosis: presentaciones clínicas infrecuentes / The case of infectious diseases: sporotrichosis: unusual clinical presentations

Se presentan dos casos de esporotricosis atípica caracterizados por lesiones duales de la piel, las membranas mucosas y los genitales externos. El diagnóstico fue comprobado por aislamiento de S. schenckii a partir de todas las lesiones; en ambos pacientes, el tratamiento antimicótico resultó en la recuperación clínica y en la negatividad de los cultivos. Estos casos ilustran la capacidad del hongo para causar lesiones en sitios insospechados, confirmando así la gama de manifestaciones clínicas de la esporotricosis.

Paracoccidioidomycosis and AIDS: report of the first two colombian cases

Relato de dois primeiros casos de pacientes colombianos com AIDS e paracoccidioidomicose. Os pacientes, ambos masculinos, nao tinham conhecimento do fator de risco por HIV, embora tivessem no passado trabalhado no campo onde poderiam ter sido infectados por fungos. Eles tiveram o tipo juvenil da doenca em varios orgaos com sintomas de curta duracao. Eles estavam profundamente imunodeprimidos, com menos de 100 CD4 T linfocitos por mL; todavia, os testes sorologicos revelaram anticorpos circulantes anti-Paracoccidioides brasiliensis em um dos pacientes e os primeiros indicios diagnosticos vieram destes testes. Em um caso, a micose precedeu o diagnostico da AIDS enquanto que no outro, ambas patologias foram descobertas simultaneamente. A terapia antimicotica com itraconazole, foi dada por 10 meses, comecando com 200mg/dia e seguida por 100mg/dia; foi notada pronunciada melhora nos sintomas e sinais da micose, sem recorrencia...