Análisis cuantitativo de la expresión de genes de resistencia a fluconazol en cepas de Candida albicans aisladas al ingreso de adultos mayores a una unidad de cuidados intensivos de Manizales, Colombia / Quantitative analysis of the expression of fluconazole-resistant genes in strains of Candida albicans isolated from elderly people at their admission in an intensive care unit in Manizales, Colombia

Introducción. Las infecciones oportunistas asociadas con Candida albicans han tenido gran repercusión en la salud pública por la mortalidad que generan en determinados grupos poblacionales. Aunque existen tratamientos farmacológicos disponibles, es evidente el aumento de la resistencia desarrollada por el agente patógeno, por lo que la determinación de los mecanismos de resistencia de las cepas presentes en las áreas hospitalarias es importante, ya que permitiría plantear mejores esquemas de tratamiento. Objetivo. Analizar la expresión de los genes ERG11, CDR1 y MDR1 en cepas de C. albicans aisladas de adultos mayores a su ingreso en la unidad de cuidados intensivos del Hospital Santa Sofía de Manizales, Colombia. Materiales y métodos. Se seleccionaron 29 muestras (21 resistentes y 8 sensibles) y se conformaron dos grupos de trabajo, uno de muestras con exposición al fluconazol y el otro sin esta. El ARN extraído se cuantificó mediante reacción en cadena de la polimerasa con transcriptasa inversa en tiempo real (RT-qPCR). Resultados. Se encontraron diferencias significativas en la expresión del gen MDR1 en el grupo de cepas de C. albicans resistentes. Dos de las cepas resistentes (104 y 62-2) expuestas al antifúngico presentaron valores muy elevados en la expresión de este gen. La expresión del ERG11 y del CDR1 no fue significativa en los grupos estudiados. Conclusión. El aumento de sobreexpresión del gen MDR1 indica que este puede ser el responsable de la resistencia; sin embargo, algunas cepas resistentes no sobreexpresaron los genes analizados, lo que indica que puede haber otros genes involucrados en la resistencia de las cepas estudiadas.

Introduction: Opportunistic infections associated with Candida albicans have had a great impact on public health due to the mortality they generate in certain population groups. Although pharmacological treatments are available, the resistance developed by the pathogen has become increasingly evident. For this reason, determining the mechanisms of resistance associated with the strains found in different hospital areas is important since it would help improving treatment plans. Objective: To analyze the expression of ERG11, CDR1, and MDR1 genes in strains of C. albicans isolated from elderly patients at admittance in the intensive care unit of Hospital Santa Sofía in Manizales, Colombia. Materials and methods: A total of 29 samples (21 resistant and 8 sensitive) were selected and distributed in two working groups: with and without exposure to fluconazole. The extracted RNA was quantified by real-time reverse transcription polymerase chain reaction (RT-qPCR). Results: Significant differences were found in the expression of the MDR1 gene in the group of resistant C. albicans strains. Two of the resistant strains (104 and 62-2) exposed to the antifungal showed very high values in the expression of this gene. The expression of ERG11 and CDR1 was not significant among the groups studied. Conclusion: The increased overexpression of the MDR1 gene indicates that it may be responsible for the resistance. However, some resistant strains did not overexpress any of the genes analyzed, which indicates that there may be other genes involved in the resistance of the strains under study.

In vitro antifungal susceptibility of clinical and environmental isolates of Aspergillus fumigatus and Aspergillus flavus in Brazil

ABSTRACT The in vitro susceptibility of 105 clinical and environmental strains of Aspergillus fumigatus and Aspergillus flavus to antifungal drugs, such as amphotericin B, azoles, and echinocandins was evaluated by the broth microdilution method proposed by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Following the EUCAST-proposed breakpoints, 20% and 25% of the clinical and environmental isolates of A. fumigatus, respectively, were found to be resistant to itraconazole (Minimal Inhibitory Concentration, MIC > 2.0 mg/L). Voriconazole showed good activity against A. fumigatus and A. flavus strains, except for one clinical strain of A. fumigatus whose MIC was 4.0 mg/L. Posaconazole (≤0.25 mg/L) also showed appreciable activity against both species of Aspergillus, except for six A. fumigatus strains with relatively higher MICs (0.5 mg/L). The MICs for Amphotericin B ranged from 0.06 to 1.0 mg/L for A. fumigatus, but were much higher (0.5-8.0 mg/L) for A. flavus. Among the echinocandins, caspofungin showed a geometric mean of 0.078 and 0.113 against the clinical and environmental strains of A. flavus, respectively, but had elevated minimal effective concentrations (MECs) for seven of the A. fumigatus strains. Anidulafungin and micafungin exhibited considerable activity against both A. fumigatus and A. flavus isolates, except for one environmental isolate of A. fumigatus that showed an MEC of 1 mg/L to micafungin. Our study proposes that a detailed investigation of the antifungal susceptibility of the genus Aspergillus from different regions of Brazil is necessary for establishing a response profile against the different classes of antifungal agents used in the treatment of aspergillosis.

Candida auris: emergence and epidemiology of a highly pathogenic yeast

Candida auris is a multidrug-resistant emerging yeast, which was responsible for healthcare-associated infection outbreaks, and was cataloged as a new species in 2009, after being isolated from a patient's ear canal secretion in Japan. Since the notification of this first occurrence, numerous cases have been reported throughout the world, including Brazil. C. auris affects mainly inpatients, patients in intensive care units, exposed to broad-spectrum antifungal medications and who make use of vascular catheters. Currently, this yeast is one of the main responsible for invasive infections in hospitals and has been cause of concern by authorities and organs due to its rapid dissemination and difficult treatment caused by its low susceptibility to antifungal agents traditionally used in clinical practice. As a contributor to the severity of infections associated with C. auris, the transmission mechanism is still unknown, which implies in a lack of control of the microorganism and high mortality rates. Thus, this literature review presents relevant information in order to alert the importance of C. auris as an etiological agent of systemic infections, as well as its epidemiology and the real challenges of the treatment (AU)

P-glycoprotein expression in oral lichen planus

Abstract: Oral lichen planus (OLP) is a stress induced inflammatory condition with malignant potency. The mdr1 (multidrug resistance) is a stress gene overexpressed in cancerous conditions and its translated form, the p-glycoprotein efflux transporter is usually overexpressed with chemotherapy, leading to chemoresistance. OLP, a lesion with carcinogenic potency, is broadly classified into the asymptomatic reticular form and the aggressive erosive form. The objective of the study was to verify the expression level of p-glycoprotein in antifungal-treated and untreated reticular OLP, in untreated erosive OLP and erosive OLP patients treated with corticosteroid. Semi-quantitative reverse transcriptase polymerase chain reaction (SQ-RTPCR) and ELISA were performed on biopsy tissue samples to evaluate the mdr1 mRNA and protein expression of p-glycoprotein, respectively. The present study shows for the first time that mdr1 mRNA as well as its translated form p-glycoprotein are overexpressed in OLP subjects compared to healthy individuals. This overexpression is significantly higher in erosive than in reticular OLP patients, further confirming that the erosive form has higher risk for multidrug resistance. A higher expression is also observed in corticosteroid-treated erosive cases than similar untreated ones. The gradation of expression is in conformity with severity of the disease.

Antibacterial activity of the Piper aduncum oil and dillapiole, its main constituent, against multidrug-resistant strains / Actividad antibacteriana del aceite de Piper aduncum y dillapiole, su componente principal, frente a cepas resistentes a múltiples fármacos

The study aimed to evaluate the bactericidal activity of oil essential and dillapiole from P. aduncum against standard and multidrug-resistant strains of Staphylococcus spp. The oil showed antimicrobial action against these strains, but better results were obtained for the standards strains of S. epidermidis and S. aureus, with MIC of 250 and 500 ug/mL, respectively. Dillapiolle was less effective than the oil against the same standard and multi-drug resistant strains (MIC =1000 ug/mL). However, when dillapiolle was tested in combination with myristicin, another component of the oil, it increased its bactericidal activity and showed a synergistic action...

El objetivo del estudio fue evaluar la actividad bactericida de los aceites esenciales y dillapiole de P. aduncum contra cepas estándar y multirresistentes de Staphylococcus spp. El aceite mostró acción antimicrobiana frente a estas cepas, pero se obtuvo mejores resultados para las cepas de S. epidermidis y S. aureus, con MIC de 250 y 500 ug/ml, respectivamente. Dillapiolle fue menos eficaz que el aceite contra cepas estándar y multirresistentes (MIC = 1000 ug/ml). Sin embargo, cuando dillapiolle fue probado en combinación con la miristicina, otro componente del aceite, que aumentó su actividad bactericida y mostró una acción sinérgica...

Is timely and appropriate antifungal drug enough for survival of adult cases with candidaemia? Five-year experience / ¿Es el uso oportuno y adecuado de antimicóticos suficiente para la supervivencia de casos de adultos con candidemia? Cinco años de experiencia

OBJECTIVE: Candidaemia is the fourth most common cause of nosocomial bloodstream infections. The objective of this paper was to evaluate the risk factors associated with mortality in patients with candidaemia with respect to Candida species and their susceptibilities, retrospectively. METHODS: All consecutive patients who developed candidaemia at an 800-bed training and research hospital were enrolled in this retrospective, observational, single centre study during the period June 2006 to December 2011. RESULTS: A total of 97 candidaemia episodes were identified in 97 patients during the study period with an overall incidence of four episodes/10 000 admissions in adults. Crude 30-day mortality rates among patients with candidaemia were 56% (55 of 97 cases). Urinary catheterization, immunosuppressive therapy, acute physiology and chronic health evaluation (APACHE) II score (>16) and hypoal-buminaemia were found to be independent risk factors for fatal candidaemia. CONCLUSIONS: Adult cases with candidaemia who have risk factors associated with mortality are more likely to have poor prognosis despite appropriate and timely initiated antifungal drug treatment. Empiric antifungal drug should be tailored according to the severity of the patients ' conditions and local antifungal susceptibility.

OBJETIVO: La candidemia es la cuarta causa más común de infecciones nosocomiales del flujo sanguíneo. El objetivo del presente trabajo fue evaluar los factores de riesgo asociados con la mortalidad en pacientes con candidemia con respecto a las especies de Candida y sus susceptibilidades, de manera retrospectiva. MÉTODOS: Todos los pacientes consecutivos que desarrollaron candidemia en un hospital de capacitación e investigación de 800 camas, fueron inscritos en este estudio retrospectivo, observacional, monocéntrico, durante el período de junio de 2006 a diciembre de 2011. RESULTADOS: Se identificaron un total de 97 episodios de candidemia en 97 pacientes durante el período de estudio con una incidencia general de cuatro episodios/10 000 ingresos en adultos. Las tasas brutas de mortalidad de 30 días entre los pacientes con candidemia fueron 56% (55 de 97 casos). Se halló que la cateterización urinaria, la terapia inmunosupresiva, y la puntuación (> 16) de la escala de Evaluación de la fisiología aguda y salud crónica (APACHE II) así como la hipoalbuminemia, constituyen factores de riesgo para una candidemia fatal. CONCLUSIONES: Los casos adultos con candidemia que tienen factores de riesgo asociados con mortalidad son más propensos a tener un pronóstico pobre a pesar del tratamiento apropiado y oportuno con medicamentos antimicóticos. Los antimicóticos empírico se deben adaptar según la severidad de las condiciones de los pacientes y la susceptibilidad antifúngica local.

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.

Species distribution and susceptibility profile of yeasts isolated from blood cultures: results of a multicenter active laboratory-based surveillance study in Argentina

The Mycology Department of the Instituto Nacional de Enfermedades Infecciosas "Dr. C. Malbrán", conducted the Second National Multicenter Survey on Fungemia due to Yeasts in Argentina. The aim was to obtain updated data of the frequency of the causative species encountered and their in vitro susceptibility to seven antifungal agents. Yeast species were identified by micromorphological and biochemical studies. Antifungal susceptibility testing was performed by the reference microdilution method E.Def 7.1 of the European Committee on Antibiotic Susceptibility Testing (EUCAST). A total of 461 viable yeasts were identified. The most frequent species were: Candida albicans (38.4 %), Candida parapsilosis (26 %), Candida tropicalis (15.4 %) and Candida glabrata (4.3 %). Other uncommon species, such as Candida viswanathii (0.6 %), Candida haemulonii (0.4 %), Candida inconspicua (0.2 %) and Candida fermentati (0.2 %) were also isolated. Among the Candida spp., 5.4 % and 1.6 % were resistant to fluconazole and voriconazole, respectively. Itraconazole and caspofungin were the most efficient agents against all Candida spp. tested (MIC < 1 mg/l). For anidulafungin, 21.6 % of C. parapsilosis showed a MIC value of 4 mg/l. Fluconazole was less active against 53.1 % of Cryptococcus neoformans (MIC > 8 mg/l), 75 % of Trichosporon spp., and 100 % of Rhodotorula spp., Geotrichum candidum, Saccharomyces cerevisiae. The global percentage of mortality was 20 %. The presence of uncommon species reinforces the need for performing continuous laboratory surveillance in order to monitor possible changes, not only in the epidemiological distribution of species, but also in the resistance to antifungal drugs.

Distribución de especies y perfil de sensibilidad de levaduras aisladas de hemocultivos: resultados de un estudio multicéntrico de vigilancia de laboratorio en Argentina. El Departamento Micología del Instituto Nacional de Enfermedades Infecciosas "Dr. Carlos G. Malbrán" condujo el segundo estudio multicéntrico nacional sobre funge- mias debidas a levaduras. El objetivo fue obtener datos actualizados sobre la distribución de especies y la sensibilidad in vitro frente a siete antifúngicos. Las levaduras fueron identificadas mediante el estudio de la micromorfología y la realización de pruebas bioquímicas. La determinación de la sensibilidad se realizó según el método de referencia E.Def 7.1 del European Committee on Antibiotic Susceptibility Testing (EUCAST). Se identificaron 461 levaduras. Las especies más frecuentes fueron Candida albicans (38,4 %), Candida parapsilosis (26 %), Candida tropicalis (15,4 %) y Candida glabrata (4,3 %). Se aislaron otras especies menos comunes, como Candida viswanathii (0,6 %), Candida haemulonii (0,4 %), Candida inconspicua (0,2 %) y Candida fermentati (0,2 %). Entre las especies del género Candida, el 5,4 % y el 1,6 % fueron resistentes al fluconazol y al voriconazol, respectivamente. El itraconazol y la caspofungina fueron los antifúngicos más eficaces in vitro frente a las especies de Candida evaluadas (CIM < 1 mg/l). Para la anidulafungina, el 21,6 % de los aislamientos de C. parapsilosis mostraron una CIM de 4 mg/l. El fluconazol fue menos activo para el 53,1 % de los aislamientos de Cryptococcus neoformans (CIM > 8 mg/l), el 75 % de los aislamientos de Trichosporon spp. y el 100 % de los aislamientos de Rhodotorula spp., Geotrichum candidum y Saccharomyces cerevisiae. El porcentaje de mortalidad fue del 20 %. La presencia de especies infrecuentes refuerza la necesidad de realizar la continua vigilancia de laboratorio con el fin de monitorear posibles cambios, no solo en la epidemiología de las especies causantes de fungemia, sino también en la resistencia a los antifúngicos.

Evaluación de tres métodos para la detección de la sensibilidad in vitro de especies de Candida a los antifúngicos / Evaluation of three methods for in vitro detection of antifungal susceptibility of Candida species

Los métodos de referencia E. Def 7.1 y M27-A3, que detectan resistencia in vitro a los antifúngicos, son onerosos y muy laboriosos, por lo que su implementación en los laboratorios hospitalarios es limitada. Existen técnicas comerciales de simple realización, que permitirían obtener resultados comparables a los que se obtienen con los métodos estándares. Los objetivos de esta investigación fueron: a) comparar los resultados de concentración inhibitoria mínima obtenidos según el método de referencia E.Def 7.1 con los obtenidos mediante el empleo del equipo comercial ATB® Fungus 3 en un conjunto de 82 aislamientos clínicos de Candida spp. frente a los siguientes antifúngicos: anfotericina B, 5-fluorocitosina, fluconazol e itraconazol; b) comparar en ese mismo conjunto de aislamientos los resultados del estudio de sensibilidad al fluconazol por difusión en agar empleando tabletas Neo-SensitabsTM o discos Malbrán con los que se obtienen por el método de referencia. La concordancia general entre el método de referencia y el ATB® Fungus 3 fue del 90,2 %, mientras que la concordancia del método de referencia con los métodos por difusión con discos y con tabletas alcanzó el 96,3% y el 92,7 %, respectivamente. El ATB® Fungus 3 fue eficaz para determinar la sensibilidad a la anfotericina B y a la 5-fluorocitosina, pero se observaron discrepancias al evaluar la sensibilidad a los azoles. Los métodos por difusión resultaron útiles para determinar la sensibilidad al fluconazol; sin embargo, observamos 3 discrepancias muy mayores, 1 mayor y 2 menores con el método de difusión con tabletas, mientras que con los discos solo se produjeron 3 discrepancias menores.

Reference methods E.Def 7.1 and M27-A3 detect in vitro resistance; however, they are expensive and very laborious. Thus, their actual use in hospital laboratories is limited. There are commercial techniques available, having easier accessibility and development, which would yield results comparable to those of the reference methods. The objectives of this study were: a) to compare the results of minimal inhibitory concentration of 82 Candida spp. clinic isolates according to reference method E.Def 7.1 and ATB® Fungus 3; b) to compare the results of fluconazole susceptibility testing by disk diffusion in agar with Neo-SensitabsTM tablets and Malbrán disks with those of the reference methods. Minimal inhibitory concentration for amphotericin B, 5-flucytosine, fluconazole and itraconazole was performed according to the E.Def 7.1 and the ATB Fungus 3 methods and diffusion was carried out with fluconazole disks and tablets. General concordance between the reference method and ATB Fungus 3 was 90.2 % and 96.3 and 92.7% for diffusion with disks and tablets. The ATB Fungus 3 method was effective to determine susceptibility against amphotericin B and 5-flucytosine; however, discrepancies were observed with azole drugs. Disk diffusion methods are useful to determine susceptibility to fluconazole; however, 3 very major, 1 major and 2 minor errors were observed with the tablets, whereas only 3 minor errors were observed with the disks.

In vitro antifungal susceptibility of Candida spp. oral isolates from HIV-positive patients and control individuals

Oropharyngeal candidiasis is the most common fungal infection among HIV-positive patients. This condition can be treated with either systemic or topical antifungal agents; treatments are usually indicated empirically on the basis of clinical data. The knowledge of in vitro antifungal susceptibility is important to determine correct therapeutic guides for the treatment of fungal infections. Therefore, the objective of this study was to determine the antifungal susceptibility profile of oral Candida isolates from HIV-positive patients and control individuals. Amphotericin B, fluconazole, flucytosine, nystatin and ketoconazole were tested according to the methodology of microdilution proposed by the Clinical and Laboratory Standards Institute (CLSI); results were recorded in values of minimal inhibitory concentration (MIC). A total of 71 Candida isolates from HIV-positive patients were examined with the following species represented: C. albicans (59), C. tropicalis (9), C. glabrata (1), C. guilliermondii (1) and C. krusei (1). A total of 15 Candida isolates were evaluated from control individuals comprised of 11 C. albicans and 4 C. tropicalis samples. Our results demonstrated that the tested antifungal agents showed good activity for most isolates from both groups; however, variability in MIC values among isolates was observed.

Bacteriocins: exploring alternatives to antibiotics in mastitis treatment

Mastitis is considered to be the most costly disease affecting the dairy industry. Management strategies involve the extensive use of antibiotics to treat and prevent this disease. Prophylactic dosages of antibiotics used in mastitis control programmes could select for strains with resistance to antibiotics. In addition, a strong drive towards reducing antibiotic residues in animal food products has lead to research in finding alternative antimicrobial agents. In this review we have focus on the pathogenesis of the mastitis in dairy cows, existing antibiotic treatments and possible alternative for application of bacteriocins from lactic acid bacteria in the treatment and prevention of this disease.

Mutants with heteroresistance to amphotericin B and fluconazole in Candida

Several studies have reported the occurrence of infections caused by Candida yeasts as well as the increasing prevalence of non albicans species. The aim of the present work is focused on the obtaining of heteroresistance to amphotericin B and fluconazole in Candida species using two distinct methodologies: selection and induction. Resistant samples were obtained by selective pressure using a medium with fluconazole for growth, followed by growth in a medium with amphotericin B. The selective pressure was also created beginning with growth in amphotericin B medium followed by growth in fluconazole medium. Concomitantly, samples were submitted to the induction of resistance through cultivation in increasing concentrations of fluconazole, followed by cultivation in increasing concentrations of amphotericin B. Subsequently, the induction began with amphotericin B followed by fluconazole. Three samples resistant to fluconazole and amphotericin B were obtained, two by induction (C. glabrata and C. tropicalis) and one by selection (C. tropicalis). Both C. tropicalis originated from the same wild sample. After successive transfers for drug free medium, only the sample obtained by selection was able to maintain the resistance phenotype. These results suggest that the phenotype of heteroresitance to fluconazole and amphotericin B can be produced by two methodologies: selection and induction.

Diagnóstico de infecção por Candida: avaliação de testes de identificação de espécies e caracterização do perfil de suscetibilidade / Candida infection diagnosis: evaluation of Candida species identification and characterization of susceptibility profile

INTRODUÇÃO: Infecções invasivas provocadas por Candida são importantes causas de morbidade e mortalidade. O sucesso do tratamento dessas infecções depende da identificação da espécie e do padrão de sensibilidade aos antifúngicos. Portanto, diagnóstico rápido e específico é fundamental para a precoce introdução de terapêutica adequada. OBJETIVOS: Avaliar diferentes métodos diagnósticos de determinação de espécies de Candida e caracterizar, entre as espécies identificadas, o padrão de sensibilidade aos diferentes antifúngicos. MATERIAL E MÉTODOS: A identificação das espécies de Candida presentes em amostras de diferentes materiais biológicos foi realizada pelo cultivo em CHROMagar® Candida e pela técnica de reação em cadeia da polimerase tipo Nested (N-PCR). O padrão de sensibilidade das amostras foi avaliado pela utilização de Etest®. RESULTADO: CHROMagar® caracterizou 50 por cento das amostras como Candida albicans; 20,8 por cento, Candida tropicalis; 2,4 por cento, Candida krusei e 26,9 por cento, outras espécies (não determinadas). As cepas de C. albicans e C. tropicalis foram caracterizadas por CHROMagar® e N-PCR. Porém cepas de outras espécies, indeterminadas em CHROMagar®, caracterizaram-se como C. parapsilosis em N-PCR. Cepas de C. krusei e C. tropicalis apresentaram perfil de resistência a, respectivamente, fluconazol e 5-fluocitosina. Quanto ao itraconazol, observou-se padrão de resistência em cepas de C. albicans e C. tropicalis. DISCUSSÃO: As técnicas metodológicas utilizadas são de fácil reprodutibilidade e alta especificidade, fornecendo diagnóstico complementar, e o emprego do Etest® viabiliza a precoce introdução de tratamento específico. CONCLUSÃO: O crescente aparecimento de espécies de Candida resistentes aos azólicos confirma a importância de monitorar possíveis mudanças na distribuição das espécies patogênicas e dos padrões de sensibilidade.

BACKGROUND: Invasive infections by Candida are an important cause of morbidity and mortality. The successful treatment of these infections depends on the identification of the species and on the sensitivity pattern to antifungal agents. A quick and specific diagnosis is essential to introduce appropriate therapy. OBJECTIVES: Assess different diagnostic methods to determine Candida species and evaluate the sensitivity pattern to different antifungal agents among the identified ones. MATERIAL AND METHODS: The identification of Candida species present in samples of different biological materials was conducted through CHROMagar® Candida culture and Nested-PCR. The sensitivity pattern was assessed using Etest®. RESULTS: The culture of samples on CHROMagar® revealed 50 percent Candida albicans, 20.8 percent Candida tropicalis, 2.4 percent Candida krusei, and 26.9 percent other undetermined species. Samples of Candida albicans and Candida tropicalis were identified through CHROMagar® and N-PCR. Species that were not determined through CHROMagar® were characterized as Candida parapsilosis by N-PCR. Resistance to fluconazole, 5-fluocytosine was detected, respectively, in Candida krusei and Candida tropicalis samples. Both Candida albicans and Candida tropicalis samples showed resistance to itraconazole. DISCUSSION: The methods applied are easily reproducible and highly specific, which allows complementary diagnosis. The determination of the susceptibility profile by Etest® enables the early introduction of specific treatment. CONCLUSION: The growing appearance of Candida species resistant to azole confirms the importance of monitoring possible changes in the distribution of pathogenic species and in the sensitivity pattern.

Candidiasis vulvovaginal en un grupo mujeres gestantes de Medellín / Vulvovaginal candidiasis in a group of pregnant women from Medellín

Introducción. En la mujer embarazada, factores como la carga hormonal y los altos niveles de glucogéno favorecen la colonización y la infección vaginal por levaduras. Objetivo. Determinar la prevalencia de levaduras del género Candida, aisladas de muestras de flujo vaginal de un grupo de mujeres embarazadas de la ciudad de Medellín y evaluar la sensibilidad a los antifúngicos. Materiales y métodos. Estudio descriptivo en el cual se estudiaron 300 mujeres gestantes que acudieron a los programas de control prenatal de diferentes IPS de Medellín, en el período de febrero de 2006 a junio de 2007. Se determinó la prevalencia de Candida spp. mediante cultivo e identificación de las levaduras obtenidas, y se determinó la sensibilidad a fluconazol e itraconazol por el método comercial del ATB fungus. A las cepas en las que se evidenció crecimiento residual en fluconazol por dicho método, se les realizó antibiograma por los métodos avalados por el Clinical and Laboratory Standards Institute (CLSI) microdilución M27-A y método de difusión en disco M-44P. Resultados. La prevalencia de Candida spp. fue de 33,3% (C. albicans, 77%; C. parapsilosis, 11%; C. tropicalis, 5%; C. glabrata, 3%; C. guillermondii, 2%; C. kefyr, 1%, y C. famata, 1%). Todos los aislamientos mostraron sensibilidad al fluconazol. Se halló resistencia al itraconazol en 9% de los aislamientos de C. albicans y en 100% de los de C. glabrata. El 2,5% de los aislamientos de C. albicans y el 100% de los de C. kefyr resultaron sensibles dependiente de la dosis a dicho antifúngico. Conclusiones. C. albicans fue la levadura más frecuentemente aislada de flujo vaginal, seguida por patógenos emergentes, como C. parapsilosis y C. tropicalis. En la población estudiada, las levaduras del género Candida son aún ampliamente sensibles a los antifúngicos. Se recomienda la identificación de la levadura a nivel de especie y hacer pruebas de sensibilidad en el caso de falla terapéutica y en infecciones recidivantes.

During pregnancy, risk factors such as the high hormonal load and high levels of glucogen allow vaginal colonization and infection by yeasts. Objective: To determine the prevalence of Candida spp. isolated from samples of vaginal discharge from pregnant women in Medellín and to test their sensitivity to antimycotic agents. Materials and methods: Descriptive study in which 300 pregnant women were tested between February 2006 to June 2007. The prevalence of Candida spp. was determined by culture, the yeasts were identified, and the sensitivity to fluconazole and itraconazole was determined by the ATB fungus method; the strains with residual growth in fluconazole by such method were submitted to antibiogram by the methods approved by the CLSI. Results: The prevalence of Candida spp. was 33.3% (C. albicans, 77%). All the isolations showed sensitivity to fluconazole. Resistance to itraconazole was found in 9% of C. albicans isolates, and in 100% of C. glabrata; 2.5% of the isolates of C. albicans and 100% of C. kefyr resulted sensitive dose-dependent to such antymicotyc. Conclusions: C. albicans was the yeast most frequently isolated from vaginal discharge, followed by emergent pathogens such as C. parasilopsis and C. tropicalis. In the studied population, yeasts from the Candida gender were still very sensitive to antimycotic agets. It is recommended to identify the yeast to its gender and to perform sensitivity tests in case of therapeutic failure or in recurrent infections.

Transporters in the Paracoccidioides brasiliensis transcriptome: insights on drug resistance

In the struggle for life, the capacity of microorganisms to synthesize and secrete toxic compounds (inhibiting competitors) plays an important role in successful survival of these species. This ability must come together with the capability of being unaffected by these same compounds. Several mechanisms are thought to avoid the toxic effects. One of them is toxin extrusion from the intracellular environment to the outside vicinity, using special transmembrane proteins, referred to as transporters. These proteins are also important for other reasons, since most of them are involved in nutrient uptake and cellular excretion. In cancer cells and in pathogens, and particularly in fungi, some of these proteins have been pointed out as responsible for an important phenotype known as multidrug resistance (MDR). In the present study, we tried to identify in the Paracoccidioides brasiliensis transcriptome, transporter-ortholog genes from the two major classes: ATP binding cassette and major facilitator superfamily transporter. We found 22 groups with good similarity with other fungal ATP binding cassette transporters, and four Paracoccidioides brasilienses assembled expressed sequence tags that probably code for major facilitator superfamily proteins. We also focused on fungicide resistance orthologs already characterized in other pathogenic fungi. We were able to find homologs to C. albicans CDR1, CDR2, and MDR1, Saccharomyces cerevisiae PDR5 and Aspergillus AtrF genes, all of them related to azole resistance. As current treatment for paracoccidioidomycosis mainly uses azole derivatives, the presence of these genes can be postulated to play a similar role in P. brasiliensis, warning us for the possibility of resistant isolate emergence.

Molecular characterization of ABC transporter-encoding genes in Aspergillus nidulans

As a preliminary step towards characterizing genes encoding ATP-binding cassette (ABC) transporters that confer pleiotropic drug resistance in Aspergillus, we used a PCR-based approach to isolate four DNA fragments corresponding to different ABC type transporter genes. DNA sequencing and Southern blot analysis confirmed that they were distinct genes, which were designated abcA-D. One of these genes, abcD, was cloned and characterized. It was found to have a predicted 1,452-amino acid translation product with a calculated molecular mass of 147,467 kDa. The abcD gene specifies a single transcript of approximately 5.0 kb; there was a two- to six-fold enhancement of mRNA levels following exposure to miconazole, camptothecin, methotrexate, and ethidium bromide

Comparación de métodos moleculares útiles en la detección rápida de Mycobacterium tuberculosis Multirresistente (MTB-MRD) / Comparation of useful molecular methods for fast detection of Multidrug Resistent Mycobacterium tuberculosis (MTB-MDR)

Objetivo: evaluar y comparar dos métodos moleculares rápidos utilizables en la detección de pacientes con MTB-MRD. Material y Métodos: realizamos un estudio experimental para comparar la eficiencia de los métodos: "rifoligotyping" e Inno-LIPA, utilizando como "gold standard" el método de las proporciones múltiples. La población de estudio fueron 41 aislamientos de pacientes con tuberculosis MRD y 43 de pacientes con tuberculosis farmacosensible. Se amplificó por PCR un fragmento del gen rpob utilizando un par de iniciadores diferentes para cada método y posteriormente se detectó mediante hibridización reversa en membranas y tiras, la presencia de mutaciones que confieren resistencia a rifampicina. Se calculó la sensibilidad, la especificidad, el valor predictivo de MRD (VPMRD) y el valor predictivo de fármaco sensibilidad (VPFS) para cada una de las pruebas y se compararon en términos de eficiencia. Resultados: las dos metodologías fueron igualmente eficientes. Los VPMRD fueron del 100 por ciento y los VPFS fueron 81.1 por ciento y 84.3 por ciento La sensibilidad fue de 75.6 por ciento y 80.5 por ciento respectivamente, y la especificidad fue de 100 por ciento para ambos métodos. Conclusiones: se pueden considerar los dos como posibles métodos de elección para determinar rápidamente la presencia de tuberculosis MRD en poblaciones de riesgo, facilitando la prevención de la diseminación de estas cepas y agilizando la prescripción de esquemas de quimioterapias apropiados. La distribución de las mutaciones demostró que se hace necesario diseñar otras membranas o tiras que contengan las mutaciones más comunes, específicamente en las cepas colombianas, porque cerca de 1/3 de los aislamientos colombianos estudiados presentaron mutaciones que no fueron identificadas por ninguno de los dos métodos

Specificity of drug transport mediated by CaMDR1: a major facilitator of Candida albicans.

CaMDR1 encodes a major facilitator superfamily (MFS) protein in Candida albicans whose expression has been linked to azole resistance and which is frequently encountered in this human pathogenic yeast. In this report we have overexpressed CaMdr1p in Sf9 insect cells and demonstrated for the first time that it can mediate methotrexate (MTX) and fluconazole (FLC) transport. MTX appeared to be a better substrate for CaMdr1p among these two tested drugs. Due to severe toxicity of these drugs to insect cells, further characterization of CaMdr1p as a drug transporter could not be done with this system. Therefore, as an alternative, CaMdr1p and Cdr1p, which is an ABC protein (ATP binding cassette) also involved in azole resistance in C. albicans, were independently expressed in a common hypersensitive host JG436 of Saccharomyces cerevisiae. This allowed a better comparison between the functionality of the two export pumps. We observed that while both FLC and MTX are effluxed by CaMdr1p, MTX appeared to be a poor substrate for Cdr1p. JG436 cells expressing Cdr1p thus conferred resistance to other antifungal drugs but remained hypersensitive to MTX. Since MTX is preferentially transported by CaMdr1p, it can be used for studying the function of this MFS protein.