Isabel Pujol

MOLECULAR DIAGNOSIS AND EPIDEMIOLOGY OF FUNGAL INFECTIONS

An experimental model of endophthalmitis by Fusarium solani in immunocompetent mice that could be useful for evaluating the efficacy of different treatments and the pathogenicity of the fungus in ocular structures was established. Five clinical isolates of F. solani were injected into the lateral tail vein of groups of 20 mice, in order to produce systemic infection with ocular infection. Inocula of 5 x 10(6) conidia per mouse were used. The eyes of the animals that died were enucleated for histopathological study to determine the degree of ocular infection. We found fungal infections in 34% of the mice studied. Panophthalmitis was detected in 16 animals, four with bilateral infections. Fungal endophthalmitis can become a severe complication of systemic mycoses by F. solani.

Experimental pathogenicity of four opportunist Fusarium species in a murine model.

A murine model with immunocompetent animals was used in a comparative study of experimental pathogenicity of 13 isolates belonging to the four most frequent pathogenic species of Fusarium in man (F. solani, F. oxysporum, F. verticillioides and F. proliferatum). Inocula of 5 x 10(6) conidia/mouse of each isolate of Fusarium were injected into a lateral vein of the tail of the mice to produce a systemic infection. F. solani was the most virulent species; the five strains of this species assayed caused the death of all the animals tested in <19 days. The other species of Fusarium were not virulent in this model. The organs mainly affected by F. solani were the kidneys and the heart. These findings correlate with the clinical evidence and demonstrate that there is a high risk associated with infection by F. solani, especially for immunocompromised patients.

Paraoxonases as Potential Antibiofilm Agents: Their Relationship with Quorum-Sensing Signals in Gram-Negative Bacteria

ABSTRACT The property of many bacteria to form biofilms constitutes a major health problem. Bacteria living in biofilms have a very high resistance to antibiotics. Biofilms may develop at a certain locations with the participation of secreted molecules, termed quorum-sensing signals, when a sufficient density of bacterial growth occurs. In Gram-negative bacteria, acyl homoserine lactones (AHL) have been identified as major quorum-sensing signals. The paraoxonases (PONs) constitute a family of enzymes comprising 3 members (PON1, PON2, and PON3) that have lactonase activity and are able to hydrolyze AHL. In this minireview, we summarize some existing basic knowledge on PON genetics, biochemistry, and function and describe recent research that reports evidence of the important roles that they may play in the organisms defense against biofilm formation. Finally, we propose some lines of future research that could be very productive.

In Vitro Interactions of Approved and Novel Drugs against Paecilomyces spp

ABSTRACT We have evaluated the in vitro activity of 15 combinations of antifungal drugs (amphotericin B, itraconazole, voriconazole, albaconazole, ravuconazole, terbinafine, and micafungin) against four isolates of Paecilomyces variotii and three of P. lilacinus. The interaction of terbinafine with the four azoles was synergistic for 53% of the combinations, while the interactions of both amphotericin B and micafungin with the rest of antifungal agents were mainly indifferent.

Comparison of Three Methods of Determining MICs for Filamentous Fungi Using Different End Point Criteria and Incubation Periods

ABSTRACT Three different methods were used to determine the in vitro activities of amphotericin B, ketoconazole, itraconazole, and flucytosine against 30 isolates of different genera of filamentous fungi. MICs were determined visually, with or without agitation, and spectrophotometrically by using a broth microdilution method. For amphotericin B, there was one end point reading criterion (the minimum concentration of antifungal that inhibited 100% of growth), but for azoles and flucytosine there were two (the minimum concentrations that inhibited 50 and 75% of fungal growth, respectively) after 48 and 72 h of incubation. All tests were performed in triplicate. An intraclass correlation coefficient (ICC) was used to evaluate the reproducibility of each of the methods and the correlation among them. The reproducibility of the three methods was very high (ICC of 0.808 to 0.992), particularly in the case of azoles and flucytosine. In general, the degree of reproducibility was highest for azoles and amphotericin B after 72 h of incubation and for flucytosine after 48 h of incubation. The degree of correlation among the three methods was very high (ICC of >0.98) with all of the antifungals under all the conditions tested. The end point reading criteria and the time of incubation affected neither the reproducibility of the methods nor their correlation, and their effect on MICs was statistically significant.

A severe case of persistent diarrhoea associated with Arcobacter cryaerophilus but attributed to Campylobacter sp. and a review of the clinical incidence of Arcobacter spp

Although rarely, Arcobacter spp. have been associated with diarrhoea and bacteraemia. We report a persistent case in a healthy 26-year-old Spanish male of bloody diarrhoea, which was attributed to Campylobacter but in fact was caused by Arcobacter cryaerophilus, as determined by sequencing of the rpoB gene. The isolate was re-identified by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) and genotyped for five putative virulence genes and for seven genes included in the Arcobacter multilocus sequence typing database. The low score obtained by MALDI-TOF indicates the need to complement the database with more isolates. Only the ciaB gene, which encodes for an invasin, was detected. Despite the isolate belonging to a new sequence type, three of the alleles (glnA, pgm and tkt) had been found previously in isolates from faeces of patients with diarrhoea. This study, together with the reviewed literature, indicates that Arcobacter can produce bacteraemia and that the isolation from patients with diarrhoea range from 0.11% to 1.25%. This study also demonstrates that Arcobacter species are confused with Campylobacter spp., as previously suggested. This is one of the factors that leads to underestimation of their incidence together with the use of inappropriate detection and identification methods.

Efficacy of Liposomal Amphotericin B in Treatment of Systemic Murine Fusariosis

ABSTRACT We have compared the activities of liposomal amphotericin B (LAMB) at 3, 5, 10, and 20 mg/kg/day and amphotericin B deoxycholate (AMB) at 1.5 and 2.5 mg/kg/day in a murine systemic infection by Fusarium verticillioides. Survival was improved by all treatments except AMB at 1.5 mg/kg/day. The tissue burden in liver was reduced by LAMB at all dosages and by AMB at 2.5 mg/kg/day. The two highest dosages of LAMB showed significant reductions in the spleen.

Use of random amplified microsatellites to type isolates from an outbreak of nosocomial aspergillosis in a general medical ward

Numerous patients were diagnosed with aspergillosis in a nosocomial outbreak caused by Aspergillus fumigatus and Aspergillus flavus. Thirty-three isolates of the former and 28 isolates of the latter were collected from the hospital environment and from the patients and studied for genetic relatedness by random amplified microsatellites (RAMS) analysis, in which two polymorphic regions were tested. Twenty-eight genotypes of A. fumigatus and 23 genotypes of A. flavus were identified. Four patients were infected by two isolates with the same genotype as the environmental isolates. One clinical genotype was shared by three patients and another was shared by two patients. We found that RAMS was useful for fingerprinting Aspergillus spp.

In Vitro and In Vivo Antifungal Susceptibilities of the Mucoralean Fungus Cunninghamella

ABSTRACT We have determined the in vitro activities of amphotericin B (AMB), voriconazole, posaconazole (PSC), itraconazole (ITC), ravuconazole, terbinafine, and caspofungin against five strains of Cunninghamella bertholletiae and four of Cunninghamella echinulata. The best activity was shown by terbinafine against both species (MIC range = 0.3 to 0.6 μg/ml) and PSC against Cunninghamella bertholletiae (MIC = 0.5 μg/ml). We have also evaluated the efficacies of PSC, ITC, and AMB in neutropenic and diabetic murine models of disseminated infection by Cunninghamella bertholletiae. PSC at 40, 60, or 80 mg/kg of body weight/day was as effective as AMB at 0.8 mg/kg/day in prolonging survival and reducing the fungal tissue burden in neutropenic mice. PSC at 80 mg/kg/day was more effective than AMB at 0.8 mg/kg/day in reducing the fungal load in brain and lung of diabetic mice. Histological studies revealed an absence of fungal elements in organs of mice treated with either AMB at 0.8 mg/kg/day or PSC at 60 or 80 mg/kg/day in both models. ITC showed limited efficacy in both models. PSC could be a therapeutic option for the treatment of systemic infections caused by Cunninghamella bertholletiae.

Antifungal Therapy in a Murine Model of Disseminated Infection by Cryptococcus gattii

ABSTRACT We have evaluated the efficacy of posaconazole (PSC), voriconazole (VRC), and amphotericin B (AMB) in a murine model of systemic infection by Cryptococcus gattii using immunocompromised animals and three clinical strains of the fungus. AMB was the most effective drug in prolonging the survival of mice and also in reducing tissue burden in all organs tested. To a lesser degree, VRC at 60 mg/kg of body weight in lung tissue and PSC at 40 mg/kg also in spleen demonstrated good efficacy in reducing the fungal load. The PSC and VRC levels in serum and brain tissue, determined by an agar diffusion bioassay method at 4 h after the last dose of the therapy, were above the corresponding MIC values. However, these drugs were not able to reduce the fungal load in brain tissue. Our results demonstrated that PSC and, to a lesser degree, VRC, have fungistatic activity and potential for the treatment of human pulmonary cryptococcosis.