Impact of ERG3 mutations and expression of ergosterol genes controlled by UPC2 and NDT80 in Candida parapsilosis azole resistance.

OBJECTIVES: Candida parapsilosis is a healthcare-related fungal pathogen particularly common among immunocompromised patients. Our understanding of antifungal resistance mechanisms in C. parapsilosis remains very limited. We previously described an azole-resistant strain of C. parapsilosis (BC014RPSC), obtained following exposure in vitro to posaconazole. Resistance was associated with overexpression of ergosterol biosynthetic genes (ERG genes), together with the transcription factors UPC2 (CPAR2-207280) and NDT80 (CPAR2-213640). The aim of this study was to identify the mechanisms underlying posaconazole resistance of the BC014RPSC strain. METHODS: To identify the causative mutation, we sequenced the genomes of the susceptible (BC014S) and resistant (BC014RPSC) isolates, using Illumina technology. Ergosterol content was assessed in both strains by mass spectrometry. UPC2 and NDT80 genes were deleted in BC014RPSC strain. Mutants were characterized regarding their azole susceptibility profile and ERG gene expression. RESULTS: One homozygous missense mutation (R135I) was found in ERG3 (CPAR2-105550) in the azole-resistant isolate. We show that Erg3 activity is completely impaired, resulting in a build up of sterol intermediates and a failure to generate ergosterol. Deleting UPC2 and NDT80 in BC014RPSC reduces the expression of ERG genes and restores susceptibility to azole drugs. CONCLUSIONS: A missense mutation in the ERG3 gene results in azole resistance and up-regulation of ERG genes expression. We propose that this mutation prevents the formation of toxic intermediates when cells are treated with azoles. Resistance can be reversed by deleting Upc2 and Ndt80 transcription factors. UPC2 plays a stronger role in C. parapsilosis azole resistance than does NDT80.

Anti-biofilm activity of low-molecular weight chitosan hydrogel against Candida species.

Candida invasive infections have increased in frequency during the last decades. Such infections are often associated to medical indwelling devices like central venous catheter. The recurrent nature and difficulties in the treatment of these infections are often related to biofilm formation. The objective of this study was to investigate the anti-biofilm activity of low-molecular weight chitosan hydrogel (LMWCH), a natural biopolymer obtained from the N-deacylation of crustacean chitin, upon clinical relevant Candida species. The in vitro ability of LMWCH to impair biofilm formation and to disorganize a preformed biofilm was tested in polystyrene microplates and quantified by the semi quantitative XTT assay and by the crystal violet assay. LMWCH in vivo efficacy as a coating for medical indwelling devices was evaluated for the first time for Candida parapsilosis, using a mouse subcutaneous foreign body model using polyurethane catheter segments. Scanning electron microscopy was used to access biofilm architecture after LMWCH treatment. We found that LMWCH efficiently impaired biofilm formation of all Candida species, also promoting biofilm disaggregation. Most importantly, LMWCH was able to significantly inhibit biofilm formation by C. parapsilosis in an in vivo catheter mouse model. SEM images showed biofilm collapsed cells compatible with membrane damage, suggesting that this could be one of the possible mechanisms underlying biofilm impairment. LMWCH revealed to be a promising compound for treatment of candidiasis or its prevention through medical device coating.

Effect of maternal deprivation on acetylcholinesterase activity and behavioral changes on the ketamine-induced animal model of schizophrenia.

Maternal deprivation has been associated with physiological and developmental changes that may be related to an increased risk for childhood and adult neuropsychiatric diseases. A growing number of studies demonstrated the importance of childhood experiences in the development of psychosis and schizophrenia in adulthood. Therefore, the present study investigated different behavior responses in rats following maternal deprivation and/or ketamine treatment in adulthood. Male rats were subjected to maternal deprivation for 180 min from postnatal day-01 to postnatal day-10. We evaluated locomotor activity, avoidance task and social interaction of adult male rats deprived or not deprived that were administered with saline or acute subanesthetic doses of ketamine (5, 15 and 25 mg/kg, i.p.). Our results show that only ketamine (25 mg/kg, i.p.) treatment in the adult rats lead to hyperlocomotion but not ketamine (5 and 15 mg/kg) and maternal deprivation alone. However, maternally deprived rats treated with ketamine (5 mg/kg) induced hyperlocomotion. Additionally, ketamine (25 mg/kg) and maternal deprivation alone induced cognitive deficit in the avoidance task. Rats deprived of and treated with ketamine (5, 15 and 25 mg/kg) also lead to memory deficit. Moreover, ketamine (25 mg/kg) and maternal deprivation alone increased latency to start social behavior. However, ketamine (5 mg/kg) and maternal deprivation lead to an increase of latency to start social behavior. Biochemistry data showed that all doses of ketamine and ketamine plus maternal deprivation increased the acetylcholinesterase (AChE) activity in the prefrontal cortex, hippocampus and striatum. The major doses of ketamine associated with maternal deprivation induced a major increase of AChE activity. Together, our results suggest that animals subjected to maternal deprivation had an increased risk for schizophrenia-like behavior and cholinergic alteration.

A novel flow cytometric protocol for assessment of yeast cell adhesion.

Microbial adhesion is a field of recognized relevance and, as such, an impressive array of tools has been developed to understand its molecular mechanisms and ultimately for its quantification. Some of the major limitations found within these methodologies concern the incubation time, the small number of cells analyzed, and the operator's subjectivity. To overcome these aspects, we have developed a quantitative method to measure yeast cells' adhesion through flow cytometry. In this methodology, a suspension of yeast cells is mixed with green fluorescent polystyrene microspheres (uncoated or coated with host proteins). Within 2 h, an adhesion profile is obtained based on two parameters: percentage and cells-microsphere population's distribution pattern. This flow cytometry protocol represents a useful tool to quantify yeast adhesion to different substrata in a large scale, providing manifold data in a speedy and informative manner.

Transcriptional profiling of azole-resistant Candida parapsilosis strains.

Herein we describe the changes in the gene expression profile of Candida parapsilosis associated with the acquisition of experimentally induced resistance to azole antifungal drugs. Three resistant strains of C. parapsilosis were obtained following prolonged in vitro exposure of a susceptible clinical isolate to constant concentrations of fluconazole, voriconazole, or posaconazole. We found that after incubation with fluconazole or voriconazole, strains became resistant to both azoles but not to posaconazole, although susceptibility to this azole decreased, whereas the strain incubated with posaconazole displayed resistance to the three azoles. The resistant strains obtained after exposure to fluconazole and to voriconazole have increased expression of the transcription factor MRR1, the major facilitator transporter MDR1, and several reductases and oxidoreductases. Interestingly, and similarly to what has been described in C. albicans, upregulation of MRR1 and MDR1 is correlated with point mutations in MRR1 in the resistant strains. The resistant strain obtained after exposure to posaconazole shows upregulation of two transcription factors (UPC2 and NDT80) and increased expression of 13 genes involved in ergosterol biosynthesis. This is the first study addressing global molecular mechanisms underlying azole resistance in C. parapsilosis; the results suggest that similarly to C. albicans, tolerance to azoles involves the activation of efflux pumps and/or increased ergosterol synthesis.

Zoonosis emergentes / Emerging zoonosis

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Celulitis de codo / Cellulitis of the elbow

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Morbilidad hospitalaria en pacientes con infección por VIH / Hospital morbidity in HIV infected patients

Fundamento y objetivo: Con el aumento de la esperanza de vida en pacientes con infección por el virus de la inmunodeficiencia humana (VIH) tras el tratamiento antirretroviral de gran actividad (TARGA), las causas de hospitalización han cambiado. Predominan ahora comorbilidades como la hepatopatía crónica, la patología cardiovascular o los efectos secundarios de los tratamientos. Nuestro objetivo es determinar las características clinicoepidemiológicas actuales de estos pacientes. Material y métodos: Estudio descriptivo y retrospectivo. Revisamos las historias clínicas de los ingresos de pacientes con infección por el VIH hospitalizados en los servicios médicos de nuestro hospital en el transcurso de un año. Resultados: Hubo 125 ingresos de 82 pacientes, un 77% varones y un 71% con infección por el virus de la hepatitis C (VHC). La edad media fue de 42 años. En el 64% la vía de transmisión fue la drogadicción parenteral. Un 36% recibían TARGA y un 51% reunían criterios sida. Los motivos de ingreso más frecuentes fueron la patologia respiratoria (32%) y las enfermedades definitorias de sida (EDS) con un 22%, de las que la tuberculosis (TBC) fue la más frecuente. Un 10% fueron ingresados en la Unidad de Cuidados Intensivos (UCI). La mortalidad fue del 11% pacientes/año. Conclusiones: La mayoría de los pacientes fueron varones, con coinfección por el VHC y usuarios de drogas vía parenteral, siendo las causas de ingreso más frecuentes las patologías respiratorias y las enfermedades oportunistas. La TBC permanece siendo la EDS más frecuente

Background and objetive: With the increasing life expectancy in human immunodeficiency virus (HIV) infected patients since the highly active antiretroviral therapy (HAART), other underlying comorbilities such as chronic liver patology, cardiovascular diseases or side effects of treatment may lead to hospitalization. The purpose of this study is to determine the clinical and epidemiological characteristics of these patients. Material and methods: A descriptive-retrospective study. We reviewed the clinical records of patients with HIV infection admitted in our medical departments through a year. Results: There were 125 hospitalizations of 82 patients. 77% were males and 71% had hepatitis C virus (HCV) infection. The median age was 42 years. 64% HIV infection had been acquired by sharing material for intravenous drug use. A 36% were receiving HAART and 51% had AIDS. The respiratory tract patology (32%) and AIDS-defining illnesses (22%) were the most frequent causes of hospitalization, and the main opportunistic disease was tuberculosis (TBC). Ten per cent were admitted in the Intensive Care Unit. Mortality rate was 11% patients/year. Conclusions: Most of the patients were male, drug users and HCV positive, and the admissions to hospital were mostly due to respiratory patology and opportunistic diseases. TBC remains like the most frequent AIDS-defining illness

[Hospital morbidity in HIV infected patients]. / Morbilidad hospitalaria en pacientes con infección por VIH.

BACKGROUND AND OBJECTIVE: With the increasing life expectancy in human immunodeficiency virus (HIV) infected patients since the highly active antiretroviral therapy (HAART), other underlying comorbilities such as chronic liver pathology, cardiovascular diseases or side effects of treatment may lead to hospitalization. The purpose of this study is to determine the clinical and epidemiological characteristics of these patients. MATERIAL AND METHODS: A descriptive-retrospective study. We reviewed the clinical records of patients with HIV infection admitted in our medical departments through a year. RESULTS: There were 125 hospitalizations of 82 patients. 77% were males and 71% had hepatitis C virus (HCV) infection. The median age was 42 years. 64% HIV infection had been acquired by sharing material for intravenous drug use. A 36% were receiving HAART and 51% had AIDS. The respiratory tract pathology (32%) and AIDS-defining illnesses (22%) were the most frequent causes of hospitalization, and the main opportunistic disease was tuberculosis (TBC). Ten per cent were admitted in the Intensive Care Unit. Mortality rate was 11% patients/year. CONCLUSIONS: Most of the patients were male, drug users and HCV positive, and the admissions to hospital were mostly due to respiratory patology and opportunistic diseases. TBC remains like the most frequent AIDS-defining illness.

Dolor abdominal y fiebre en paciente anticoagulado / Dolor and selective IgA deficiency

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