The objective of this study was to determine whether seeds of
Brassica oleracea
var
. italica
(i.e. broccoli, an edible plant) produce defensins that inhibit phytopathogenic fungi and pathogenic ...bacteria of clinical significance. Crude extracts obtained from broccoli seeds were fractioned by molecular exclusion techniques and analyzed by liquid chromatography-high-resolution mass spectrometry. Two peptides were identified, BraDef1 (10.68 kDa) and BraDef2 (9.9 kDa), which were categorized as Class I defensins based on (a) their primary structure, (b) the presence of four putative cysteine disulfide bridges, and (c) molecular modeling predictions. BraDef1 and BraDef2 show identities of, respectively, 98 and 71%, and 67 and 85%, with defensins from
Brassica napus
and
Arabidopsis thaliana
. BraDef (BraDef1 + BraDef2) disrupted membranes of
Colletotrichum gloeosporioides
and
Alternaria alternata
and also reduced hyphal growth of
C. gloeosporioides
by ~ 56% after 120 h of incubation. Pathogenic bacteria (
Bacillus
cereus
183,
Listeria monocytogenes
,
Salmonella typhimurium
,
Pseudomonas aeruginosa
, and
Vibrio parahaemolitycus
) were susceptible to BraDef, but probiotic bacteria such as
Bifidobacterium animalis
,
Lactobacillus acidophilus
, and
Lactobacillus casei
were not inhibited
.
To our knowledge, this is the first report of defensins present in seeds of
B. oleracea
var.
italica
(i.e. edible broccoli). Our findings suggest an applied value for BraDef1/BraDef2 in controlling phytopathogenic fungi and pathogenic bacteria of clinical significance.
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.
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.
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.
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.
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.
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.
We compared different genotyping markers in their assessment of the genetic variability and molecular identification of clinical isolates of Fusarium.
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.
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.
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.
En la literatura colombiana son escasos los reportes acerca de la epidemiología de la tinea capitis.
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.
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.
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%).
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.
There are few written reports on the epidemiology of tinea capitis in Colombia.
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.
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.
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%).
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.
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.
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.
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.
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.
The aim of the present study was to evaluate the antifungal susceptibilities of isolates of Fusarium to amphotericin B, itraconazole and voriconazole.
The susceptibility of 44 isolates of Fusarium ...was tested by the E-test methodology.
All the isolates were resistant to itraconazole, and 89 % and 54,5 % were resistant to amphotericin B and voriconazole, respectively.
The results confirm the high level of resistance reported, regardless of the species or the strain of Fusarium involved. The high MICs level observed are worrying and suggest that new drugs are needed.
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