Microbial Warfare on Three Fronts: Mixed Biofilm of Aspergillus fumigatus and Staphylococcus aureus on Primary Cultures of Human Limbo-Corneal Fibroblasts.

Background: Coinfections with fungi and bacteria in ocular pathologies are increasing at an alarming rate. Two of the main etiologic agents of infections on the corneal surface, such as Aspergillus fumigatus and Staphylococcus aureus, can form a biofilm. However, mixed fungal-bacterial biofilms are rarely reported in ocular infections. The implementation of cell cultures as a study model related to biofilm microbial keratitis will allow understanding the pathogenesis in the cornea. The cornea maintains a pathogen-free ocular surface in which human limbo-corneal fibroblast cells are part of its cell regeneration process. There are no reports of biofilm formation assays on limbo-corneal fibroblasts, as well as their behavior with a polymicrobial infection. Objective: To determine the capacity of biofilm formation during this fungal-bacterial interaction on primary limbo-corneal fibroblast monolayers. Results: The biofilm on the limbo-corneal fibroblast culture was analyzed by assessing biomass production and determining metabolic activity. Furthermore, the mixed biofilm effect on this cell culture was observed with several microscopy techniques. The single and mixed biofilm was higher on the limbo-corneal fibroblast monolayer than on abiotic surfaces. The A. fumigatus biofilm on the human limbo-corneal fibroblast culture showed a considerable decrease compared to the S. aureus biofilm on the limbo-corneal fibroblast monolayer. Moreover, the mixed biofilm had a lower density than that of the single biofilm. Antibiosis between A. fumigatus and S. aureus persisted during the challenge to limbo-corneal fibroblasts, but it seems that the fungus was more effectively inhibited. Conclusion: This is the first report of mixed fungal-bacterial biofilm production and morphological characterization on the limbo-corneal fibroblast monolayer. Three antibiosis behaviors were observed between fungi, bacteria, and limbo-corneal fibroblasts. The mycophagy effect over A. fumigatus by S. aureus was exacerbated on the limbo-corneal fibroblast monolayer. During fungal-bacterial interactions, it appears that limbo-corneal fibroblasts showed some phagocytic activity, demonstrating tripartite relationships during coinfection.

[Molecular markers: an important tool in the diagnosis, treatment and epidemiology of invasive aspergillosis]. / Marcadores moleculares: una herramienta importante en el diagnóstico, tratamiento y epidemiología de la aspergilosis invasora.

Increase in the incidence of invasive aspergillosis has represented a difficult problem for management of patients with this infection due to its high rate of mortality, limited knowledge concerning its diagnosis, and therapeutic practice. The difficulty in management of patients with aspergillosis initiates with detection of the fungus in the specimens of immunosuppressed patients infected with Aspergillus fumigatus; in addition, difficulty exists in terms of the development of resistance to antifungals as a consequence of their indiscriminate use in prophylactic and therapeutic practice and to ignorance concerning the epidemiological data of aspergillosis. With the aim of resolving these problems, molecular markers is employed at present with specific and accurate results. However, in Mexico, the use of molecular markers has not yet been implemented in the routine of intrahospital laboratories; despite the fact that these molecular markers has been widely referred in the literature, it is necessary for it to validated and standardized to ensure that the results obtained in any laboratory would be reliable and comparable. In the present review, we present an update on the usefulness of molecular markers in accurate identification of A. fumigatus, detection of resistance to antifugal triazoles, and epidemiological studies for establishing the necessary measures for prevention and control of aspergillosis.

El incremento en la incidencia de la aspergilosis invasora representa un grave problema para el tratamiento de pacientes con esta micosis, debido a su elevada tasa de mortalidad por deficiencias diagnósticas y terapéuticas. Éstas se han atribuido a la dificultad para detectar Aspergillus fumigatus, principal agente etiológico de esta micosis, en las muestras biológicas de pacientes inmunosuprimidos, que son los principales afectados por el hongo; además por la resistencia a los antifúngicos como consecuencia del uso incontrolado de éstos, a nivel profiláctico y terapéutico, y el desconocimiento de aspectos epidemiológicos de la aspergilosis. En la actualidad, para superar estas limitaciones se han empleado marcadores moleculares. En México su uso aún no está implementado en la rutina de los laboratorios intrahospitalarios, porque a pesar de que se han reportado ampliamente en la bibliografía, hace falta validarlos y estandarizarlos para asegurar que los resultados que se obtengan en cualquier laboratorio sean confiables y comparables. En este trabajo se presenta una revisión actualizada de la utilidad de los marcadores moleculares en la identificación certera de A. fumigatus en la detección de resistencia a los antifúngicos triazólicos y en estudios epidemiológicos para establecer las medidas necesarias en la prevención y control de la aspergilosis.

Exploratory study on the clinical and mycological effectiveness of a herbal medicinal product from Solanum chrysotrichum in patients with Candida yeast-associated vaginal infection.

Mexican traditional medicine uses Solanum chrysotrichum to treat fungi-associated dermal and mucosal illness; its methanolic extract is active against dermatophytes and yeasts. Different spirostanic saponins (SC-2-SC-6) were identified as the active molecules; SC-2 was the most active in demonstrating a fungicidal effect against Candida albicans and non-albicans strains. The aim of the present study was to compare the clinical (elimination of signs and symptoms) and mycological effectiveness (negative mycological studies) of an S. chrysotrichum herbal medicinal product (Sc-hmp), standardized in 1.89 mg of SC-2, against ketoconazole (400 mg) in the topical treatment of cervical and/or vaginal infection by Candida. Both treatments (vaginal suppositories) were administered daily during 7 continuous nights. The study included 101 women (49 in the experimental group) with a confirmed clinical condition and positive mycological studies (direct examination and/or culture) of Candida infection. Basal conditions did not show differences between the groups; a moderate clinical picture was present in 62% of the cases, direct examination was positive in 69%, and the culture was positive with C. albicans predominating (65%). At the end of the administration period, both treatments demonstrated 100% tolerability, and clinical cure in 57.14% of S. chrysotrichum-treated cases and in 72.5% of ketoconazole-treated cases (p = 0.16), as well as 62.8% and 97.5% of mycological effectiveness, respectively (p = 0.0 001). We conclude that, at the doses used, Sc-hmp exhibits the same clinical effectiveness as ketoconazole, but with lower percentages of mycological eradication. Additional clinical studies with Sc-hmp are necessary, with increasing doses of SC-2, for improving the clinical and mycological effectiveness.

Mycological and electron microscopic study of Solanum chrysotrichum saponin SC-2 antifungal activity on Candida species of medical significance.

Solanum chrysotrichum is utilized in traditional Mexican medicine for the treatment of mycotic skin infections. Several microbiological studies have provided evidence of its antifungal activity against dermatophytes and yeasts. S. chrysotrichum saponins have been identified as a group of compounds with antifungal activity and saponin SC-2 has demonstrated to be the most active. Previous clinical studies have shown the therapeutic effectiveness of S. chrysotrichum-derived saponin-standardized herbal products in the treatment of Tinea pedis and Pityriasis capitis. There is no previous evidence of the activity of these saponins against Candida non-albicans species, or fluconazole- and ketoconazole-resistant Candida strains. The present study reports the biological activity of the SC-2 saponin (inhibitory concentration [IC (50)] and minimum fungicide concentration [MFC]), against 12 Candida strains of clinical significance ( C. albicans, five strains; C. glabrata and C. parapsilosis, two; C. krusei, C. lusitaniae and C. tropicalis, one), including some fluconazole (Fluco)- and ketoconazole (Keto)-resistant clinical isolates. In addition, SC-2-associated microstructural alterations were reported in four of the above-mentioned Candida species. Seven strains had IC (50) of 200 microg/mL for SC-2, 400 microg/mL was found in four strains, and 800 microg/mL for a sole C. glabrata strain. Susceptibility to SC-2 saponin was as follows: C. albicans = C. lusitaniae > C. krusei > C. glabrata. The MFC was 800 microg/mL for the majority of strains (nine), 400 microg/mL for C. albicans (two strains) and C. lusitaniae. The ultrastructural Candida changes originated by SC-2 included the following: 1) damage on cytoplasmic membrane and organelles; 2) changes in cell wall morphology and density, with separation of cytoplasmatic membrane from cell wall and disintegration of the latter; and 3) total degradation of cellular components and death. Changes were manifested from 6 h of incubation, reaching their maximum effect at 48 h. In conclusion, the saponin SC-2 possesses fungicide and fungistatic activity on different Candida albicans and non- albicans species (including some azole-resistant strains) with IC (50) values of 200 microg/mL (in Fluco-susceptible strains) and of 400 - 800 mug/mL (in Fluco-resistant strains). Additionally, we observed by transmission electron microscopy (TEM) that saponin SC-2 causes severe changes in all fungal cell membranes, and to a lesser degree on the cell wall.

Clinical and mycological evaluation of therapeutic effectiveness of Solanum chrysotrichum standardized extract on patients with Pityriasis capitis (dandruff). A double blind and randomized clinical trial controlled with ketoconazole.

Dandruff (also called Pityriasis capitis) is a seborrhoeic dermatitis of the scalp. It has been correlated with the pathological colonization of the scalp with yeast from the genus Malassezia; this illness has a worldwide distribution and represents 25% of all scalp dermatosis cases. It has been demonstrated that the extract obtained from leaves of the plant Solanum chrysotrichum possesses biological activity against dermatophytes and yeast. Different steroidal saponins with antimycotic activity have been isolated from the active extract. Clinical trials with standardized extracts prepared with this vegetal species report high rates of clinical and mycological effectiveness in the treatment of Tinea pedis,without producing secondary effects. The aim of this randomized, double blind and controlled clinical study, was to compare the therapeutic effectiveness and tolerability of a shampoo containing a standardized extract of S. chrysotrichum (applied every third day, for 4 weeks), against 2% ketoconazole in the topical treatment of Pityriasis capitis. From a total of 120 patients with the clinical diagnosis of Pityriasis capitis, 14 subjects were eliminated because the presence of Malassezia was not proved, an-other two patients withdrew from the study due to non-medical causes and one more withdrew because Tinea capitis was diagnosed. Therefore, the final analysis included 51 subjects in the experimental group and 52 in the control; in 45.6% of the cases M. furfur was identified as the pathogenic agent, in 44.66% M. globosa was isolated, and 9.71 % of the patients had a mixed infestation. At the end of the treatment period, the prepared phytopharmaceutical with the standardized extract from S. chrysotrichum achieved a clinical effectiveness (total absence of signs and symptoms produced by Pityriasis capitis) of 92.16%;the mycological effectiveness (absence of Malassezia spp. in the direct examination and culture) was 68.63 %; whilst the tolerability (absence of side effects that prompt subjects to abandon the treatment) was 100%. The therapeutic success (clinical and mycological effectiveness plus tolerability) was 64.71%. The comparison of these results with that obtained from the group treated with 2% ketoconazole, showed no significant differences (Z2, p >0.23). These results show the therapeutic effectiveness and tolerability of the standardized extract from S. chrysotrichum on the local treatment of Pityriasis capitis associated with the yeast of the genus Malassezia.

Tipificación de aislados clínicos de Histoplasma capsulatum por métodos fenotípicos y genotípicos / Typing of Histoplasma capsulatum clinical isolates by phenotype and genotype methods

Objetivo: El presente trabajo plantea el estudio fenotípico y genotípico de cinco aislados de Histoplasma capsulatum obtenidos de pacientes mexicanos con hostoplasmosis asociada a síndrome de inmunodeficiencia adquirida (SIDA), con procedencia geográfica bien establecida. Material y métodos: Se utilizaron para relacionar estos aislados clínicos, electrofóresis en geles de poliacrilamida dodecil sulfato de sodio (SDS-PAGE) y electroinmunotransferencia (EIT) para la fenotipificacion, y el polimorfismo del DNA amplificado al azar por la reacción en cadena de la polimerasa (RAPD-PCR) para la genotificación. Las relaciones enter los patrones electoforéticos y polimórficos de todos los aislado se procesaron en el programa SPSS/PC+ versión 2.3. Resultados y discusión: Por SDS-PAGE se observó que todos los aislados se relacionaron en un 86 por ciento. Por EIT se evidenció que todos los aislados revelaron tres bandas de 43, 28 y 18 kDa con suero inmune específico. Por RAPD-PCR, cuatro aislados se relacionaro en 94 por ciento y el último (EH-319) presentó 77 por ciento de relación con los anteriores. Conclusión: El análisis por RAPD.PCR permitió discriminar ligeras diferencias entre los aislados de pacientes mexicanos con histoplasmosis asociada a SIDA, en comparación con los métodos de caracterizacion fenotípica (SDS-PAGE y EIT)

Genotipificación de cepas de Histoplasma Capsulatum aisladas de pacientes con histoplasmosis asociada al SIDA, mediante el polimorfismo en la longitud de los fragmentos de restricción / Genotypic characterization of Histoplasma capsulatum isolates from AIDS associated-histoplasmosis-patients by using restriction fragment langth polymorphysms (RFLP)

Introducción: La histoplasmosis es una de las más importantes micosis asociada al síndrome de inmunodeficiencia adquirida (SIDA) en México. En este trabajo se efectuó la caracterización genotípica por el polimorfismo en la longitud de los fragmentos de restricción (RFLP) del DNA total de 31 cepas de Histoplasma capsulatum de pacientes con histoplasmosis asociada a SIDA, y su comparación con cepas de referencia de México, Panamá y Estados Unidos de America (EUA). Material y métodos: Las cepas fueron plenamente identificadas como H. capsulatum mediante su morfología colonial y microscópica, conversión de micelio a levadura y repoducción de exoantígenos probados por inmunodifusión con sueros específicos. El DNA total de las cepas fue obtenido a partir de micelio a levadura y producción de exoantígenos probados por inmunodifusión con sueros específicos. El DNA total de las cepas fue obtenido a partir de micelio macerado con nitrógeno líquido e incubado en presencia de proteinasa K y dodecilsulfato de sodio. El DNA fue diferido con la enzima HaeIII y los productos separados por electroforesis en agarosa y teñidos con bromuro de etidio. Utilizando el programa SPSS/PC+ se construyó un dendrograma a fin de establecer las relaciones polimórficas entre las cepas. Resultados y discusión: Las cepas formaron 10 grupos según sus perfiles de RFLP, obsevándose dos perfiles principales A y C con 33.3 y 29.1 por ciento de las cepas, respectivamente. Los perfiles restantes estuvieron representados por cepas únicas, a excepción del perfil J con dos cepas que mostraron un patrón similar al de la cepa Downs (clase 1, de EUA), No se encontró ningún perfil semejante a los de las cepas prevalentes en EUA o Panamá. El dendrograma mostró una relación de 85 por ciento entre las cepas de los perfiles de mayor prevalencia (A y C) asimismo del perfil J, además de las cepas Downs y FLs1 de los EUA. Los demás aislados con perfiles únicos se relacionaron en un 75 por ciento con las otras cepas

Histoplasmosis en México, aspectos históricos y epidemiológicos / Histoplasmosis in Mexico, historic and epidemiological aspects

Introducción: La histoplasmosis es una enfermedad pulmonar considerada como ocupacional. Su agente etiológico. Histoplasma capsulatum, es un hongo dimórfico distribuido en sitios donde existen cantidades importantes de excretas de murciélagos y aves. Los brotes epidémicos son frecuentemente originados por las visitas hacia los ambientes cerrados por alguna actividad laboral o recreativa. Este estudio informa sobre la distribución de la histoplasmosis en México. Se conformó con las publicaciones que se han hecho hasta la fecha, muchas de las cuales presentan datos poco sistematizados sobre la enfermedad en el país, obtenidos con diferentes metodologías y en diversas circunstancias. Aspectos históricos y estado actual de la histoplasmosis en México: Se contemplan aspectos históricos fundamentales, así como los epidemiológicos, que permiten conjuntar un marco del conocimiento acutal de la histoplasmosis en el país. Se rescatan datos que indican que la histoplasmosis en México data desde 1895 y se presentan información reciente que sugiere que la enfermedad está ampliamente distribuida en la mayoría de los estados de la República, predominando en los estados del sur y centro, generalmente ocasionada por brotes importantes, con mayor frecuencia en individuos mayores de 15 años. La importancia de este estudio es el advertir a las autoridades de salud sobre la actual prevalencia y distribución de la enfermedad en el país y proponer mecanismos para su reincorporación al Sistema Nacional de Vigilancia Epidemiológica así como criterios para la asignación de su endemicidad en el país