Fongs oportunistes: Avaluació de la patogènia i sensibilitat als antifungics

Les infeccions greus causades per fongs han augmentat molt en els últims anys, i sobretot les causades pels anomenats fongs oportunistes. Aquests últims acostumen a provocar infeccions en pacients que tenen altres malalties de base, com per exemple leucèmia, SIDA, càncer, o han rebut trasplantaments d'òrgans sòlids o de moll d'os. La llista de les espècies de fongs oportunistes implicades en infeccions greus és cada dia més llarga, i les estratègies per tractar aquestes infeccions continuen essent molt limitades. Els tres gèneres de fongs filamentosos oportunistes que han estat objecte d'estudi a la present tesi, Fusarium, Scedosporium i Paecilomyces es troben dins d'aquest grup de fongs oportunistes. Encara que només provoquen infeccions greus en pacients immunocompromesos, la taxa de mortalitat d'aquestes infeccions s'acosta al 100%. L'amfotericina B ha estat durant molts anys, i encara ho és en l'actualitat, el fàrmac més utilitzat per tractar les infeccions sistèmiques, però té importants problemes de toxicitat. En els últims anys s'han desenvolupat altres formulacions d'aquest mateix fàrmac, que presenten molts menys problemes de toxicitat. L'objectiu principal d'aquesta tesi ha estat avaluar l'eficàcia d'una de les noves formulacions de l'amfotericina B, el preparat liposòmic, en models animals d'infecció disseminada.Per poder comparar els diferents tractaments primer ha calgut desenvolupar models animals d'infecció per cadascun dels fongs estudiats (utilitzant ratolins), que siguin reproduïbles i que puguin simular la infecció fúngica humana.Un cop desenvolupats els models animals es va comparar l'eficàcia de les dues formulacions de l'amfotericina B, en el tractament de les infeccions causades pels fongs estudiats. Això s'avaluava comparant amb mètodes estadístics el temps de supervivència (TMS) dels diferents grups. També es comparava la quantitat de fong present a diferents òrgans dels ratolins, que eren sacrificats quan finalitzava el tractament. A part de l'amfotericina B que és, tal i com hem dit, el fàrmac més utilitzat per combatre les infeccions provocades per fongs oportunistes, hi ha d'altres antifúngics, però han demostrat poca eficàcia al menys in vitro, i els resultats clínics són molt escassos. És per això que hem volgut avaluar el possible efecte sinèrgic d'algunes combinacions d'antifúngics in vitro.De forma resumida, les conclusions principals que es desprenen dels diversos estudis realitzats són els següents.1- Es van establir models d'infecció disseminada en ratolins per les espècies següents Paecilomyces variotii, P. lilacinus, P. javanicus, Fusarium verticillioides, Scedosporium prolificans, S. apiospermum en ratolins immunodeprimits o immunocompetents.2- Es va avaluar l'eficàcia de les dues formulacions de l'antifúngic amfotericina B (el desoxicolat i la formulació liposòmica) en diferents models d'infecció experimental desenvolupats en la primara fase dels estudis.En ratolins infectats amb P. variotii, les dues formulacions van ser equivalents a dosis baixes, però l'administració d'amfotericina B liposòmica a 10 mg/kg va provocar una disminució significativa de la presència del fong en alguns òrgans.Les dosis baixes d'amfotericina B liposòmica va fer augmentar el TMS dels ratolins infectats amb F. verticillioides. Les dosis altes, a més, van fer disminuir significativament la presència del fong en diversos òrgans.En els ratolins infectats per S. prolificans, es va observar un increment del TMS, que va ser més evident amb la dosi més alta d'amfotericina B liposòmica. L'administració de G-CSF amb la teràpia antifúngica no va millorar els resultats. 3- S'ha avaluat l'activitat in vitro de nou combinacions d'antifúngics davant de diverses espècies dels gèneres Fusarium i Paecilomyces.Les combinacions que s'han comportat de forma sinèrgica per un nombre més elevat de soques han estat les de ravuconazol combinat tant amb amfotericina B com amb terbinafina, i aquesta última combinada amb voriconazol. Les combinacions que s'han comportat de forma sinèrgica per un nombre més elevat de soques han estat totes les de terbinafina combinada amb azols.Deep infections caused by moulds have increased during the last years, and especially those caused by opportunistic fungi. These fungi use to provoke infections in patients with underlying diseases such as leukaemia, AIDS, cancer, and solid organ or bone marrow transplant recipients. The number of fungal species involved in deep infections increases every day, and the strategies to treat these infections are still limited. The three genera of filamentous fungi that have been studied in the present thesis Fusarium, Scedosporium and Paecilomyces are included in the group of emerging opportunistic pathogens. Even though they just provoke severe infections in immunocompromised patients, the mortality rate is close to 100%. Amphotericin B has been for many years, and is still nowadays, the most used drug to treat the systemic infections, but it has important toxicity problems. In recent years newer formulations of the drug have been developed, which show less toxicity problems. The main objective of the present thesis has been to evaluate the efficacy of one of the newer formulations of amphotericin B, the liposomal preparation, in murine models of disseminated infection.To be able to compare both treatments we previously developed reproducible animal models of infection (using mice) that could simulate the human infection, for each one of the strains. This was in order to compare the efficacy of both formulations of amphotericin B, in the treatment of the infections caused by the studied fungi. This was evaluated by comparing using statistic methods the mean survival time of the different groups of mice (MST). We also compared the tissue burden of the fungi in several target organs of mice, which were sacrificed after the treatment. In addition to amphotericin B which is, as we said before, the most widely used drug to treat infections caused by opportunistic fungi, there are other antifungal agents, but they have shown low efficacy, at least in vitro, and the clinical results are very scarce. That is why we wanted to evaluate the possible synergic effect in vitro of some of these drug combinations.In a resumed way, the main conclusions derived from the several performed studies are as follows:1- We established murine models of disseminaded infection by the following species: Paecilomyces variotii, P. lilacinus, P. javanicus, Fusarium verticillioides, Scedosporium prolificans, and S. apiospermum in immunosuppressed or immunocompetent mice.2- We evaluated the efficacy of two formulations of the antifungal agent amphotericin B (the deoxycholate and the liposomal formulations) in the different experimental infection models developed in the first phase of the studies.In mice infected with P. variotii, both formulations showed similar results at low doses, but the administration of liposomal amphotericin B at 10 mg/kg provoked a significant decrease of the presence of the fungus in target organs.Low doses of liposomal amphotericin B increased the MST of mice infected with F. verticillioides. At high doses, it also lowered the presence of the fungus in several organs.In mice infected with S. prolificans, the administration of high doses of high doses of liposomal amphotericin B provoked an increase of MST, which was more evident with the highest dose. The administration of G-CSF together with the antifungal therapy did not improve the results. 3- We evaluated the in vitro activity of nine combinations of antifungal drugs against several species of the genera Fusarium and Paecilomyces.The combinations that showed a highest percentage of synergic effects for Fusarium were ravuconazole combined with amphotericin B or terbinafine, and terbinafine with voriconazole. The combinations that showed a highest percentage of synergic effects for Paecilomyces were terbinafine combined with any of the azoles

Differential expression of genes involved in iron metabolism in Aspergillus fumigatus

The ability of fungi to survive in many environments is linked to their capacity to acquire essential nutrients. Iron is generally complexed and available in very limited amounts. Like bacteria, fungi have evolved highly specific systems for iron acquisition. Production and uptake of iron-chelating siderophores has been shown to be important for certain human bacterial pathogens, as well as in fungal pathogens such as Cryptococcus neoformans and Fusarium graminearum. This system also enables the opportunistic fungal pathogen Aspergillus fumigatus to infect and subsequently colonize the human lung. In this study, advantage was taken of genome sequence data available for both Aspergillus nidulans and A. fumigatus either to partially clone or to design PCR primers for 10 genes putatively involved in siderophore biosynthesis or uptake in A. fumigatus. The expression of these genes was then monitored by semi-quantitative and quantitative real-time PCR over a range of iron concentrations. As expected, the putative biosynthetic genes sidA, sidC and sidD were all strongly up-regulated under iron starvation conditions, although the variable degree of induction indicates complex regulation by a number of transcriptional factors, including the GATA family protein SreA. In contrast, the gene sidE shows no iron-regulation, suggesting that SidE may not be involved in siderophore biosynthesis. The characterisation of the expression patterns of this subset of genes in the iron regulon facilitates further studies into the importance of iron acquisition for pathogenesis of A. fumigatus. [Int Microbiol 2006; 9(4):281-287

Removal of phosphate from River water using a new baffle plates electrochemical reactor

During the last 50 years, the human activities have significantly altered the natural cycle of phosphate in this planet, causing phosphate to accumulate in the freshwater ecosystems of some countries to at least 75% greater than preindustrial levels, which indicates an urgent need to develop efficient phosphate treatment methods. Therefore, the current study investigates the removal of phosphate from river water using a new electrochemical cell (PBPR). This new cell utilises perforated baffle plates as a water mixer rather than magnetic stirrers that require power to work. This study investigates the influence of key operational parameters such as initial pH (ipH), current density (Ј), inter-electrode distance (ID), detention time (t) and initial phosphate concentration (IC) on the removal efficiency, and influence of the electrocoagulation process on the morphology of the surface of electrodes. Overall, the results showed that the new reactor was efficient enough to reduce the concentration of phosphate to the permissible limits. Additionally, SEM images showed that the Al anode became rough and nonuniform due to the production of aluminium hydroxides. The main advantages of the electrocoagulation technique are: 1- The EC method does not produce secondary pollutants as it does not required chemical additives, while other traditional treatment methods required either chemical or biological additives [[1], [2], [3], [4]]. 2- It has a large treatment capacity and a relatively short treatment time in comparison with other treatment methods, such as the biological methods [1,[5], [6], [7]]. 3- The EC method produces less sludge than traditional treatment traditional chemical and biological treatment methods [8,9]. EC technology, like any other treatment method, has some drawbacks that could limit its performance. For instance, it still has a clear deficiency in the variety of reactor design, and the electrodes should be periodically replaced as they dissolve into the solution due to the oxidation process [2,10]

Electrocoagulation as a green technology for phosphate removal from River water

The current study investigates the removal of phosphate from water using a new baffle plates aluminium-based electrochemical cell (PBPR) taking consideration the influence of key operating parameters. This new cell utilises perforated baffle plates as a water mixer rather than magnetic stirrers that require extra power to work. As this unit is new, a comprehensive study has been carried to assess it performance. This study also includes preliminary estimates of the reactor’s operating costs, the amount of H2 gas produced and the yieldable energy from it. SEM (scanning electron microscope) was used to investigate the influence of the electrocoagulation process on the morphology of the surface of aluminium electrodes, and an empirical model developed to reproduce the phosphate removal process. The results showed that 99% of phosphate was removed within 60 minutes of electrolysis at an initial pH (ipH) of 6, inter-electrode distance (ID) of 0.5 cm, current density (J) of 6 mA/cm2, initial concentration of phosphate (IC) of 100 mg/L, and minimum operating cost of 0.503 US $/m3. The electrochemical cell produced enough H2 gas to generate 4.34 kWh/m3 of power. Statistically, it was proved that the influence of the operating parameters on phosphate removal could be modelled with an R2 of 0.882, the influence of these operating parameters on phosphate removal following the order: t>J>IC>ipH >ID. Finally, SEM images showed that after several electrolysing runs, the Al anode became rough and nonuniform which could be related to the production of aluminium hydroxides

Efficacy of Liposomal Amphotericin B in Treatment of Systemic Murine Fusariosis

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

In Vitro Antifungal Susceptibilities of Uncommon Basidiomycetous Yeasts

The in vitro activities of eight antifungal drugs against 50 isolates of basidiomycetous yeasts were determined by a microdilution method. In general fluconazole and micafungin were inactive. Terbinafine was active only against Sporobolomyces salmonicolor. The activities of the other antifungals were variable and depended on the species tested. The new triazoles showed the lowest MICs, but amphotericin B and itraconazole were the only drugs active against Cryptococcus albidus

Efficacy of Albaconazole (UR-9825) in Treatment of Disseminated Scedosporium prolificans Infection in Rabbits

There are no effective therapeutics for treating invasive Scedosporium prolificans infections. Doses of 15, 25, and 50 mg/kg of body weight/day for the new triazole albaconazole (ABC) were evaluated in an immunocompetent rabbit model of systemic infection with this mold. Treatments were begun 1 day after challenge and given for 10 days. ABC at any dose was more effective than amphotericin B (AMB) at 0.8 mg/kg/day at clearing S. prolificans from tissue (P < 0.007). The percentages of survival at 25 mg of ABC/kg/day were similar to those obtained with AMB. Rabbits showed 100% survival when they were treated with 50 mg of ABC per kg (P < 0.0001 versus control group), and only this dosage was able to reduce tissue burden significantly in the five organs studied, i.e., spleen, kidneys, liver, lungs, and brain

In Vitro Interactions of Approved and Novel Drugs against Paecilomyces spp.

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

In Vitro Activities of New Antifungal Agents against Chaetomium spp. and Inoculum Standardization

Chaetomium is an unusual etiological agent of human infections, but the mortality rate among immunocompromised patients is considerably greater than that among nonimmunocompromised individuals. We investigated the in vitro antifungal susceptibilities to novel antifungal agents of 19 strains belonging to three species of Chaetomium which have been involved in human infections, i.e., Chaetomium globosum, C. atrobrunneum, and C. nigricolor, and one strain of the closely related species Achaetomium strumarium. A modification of the NCCLS reference microdilution method (M38-A) was used to evaluate the in vitro activities of ravuconazole, voriconazole, albaconazole, and micafungin. Micafungin was not active at all, while the geometric mean MICs and minimum effective concentrations of the three triazoles were less than 0.5 and 0.4 μg/ml, respectively