An alternative LMI static output feedback control design for discrete-time nonlinear systems represented by Takagi-Sugeno models.

This paper presents a static output feedback controller design for discrete-time nonlinear systems exactly represented by Takagi-Sugeno models. By introducing past states in the control law as well as in the Lyapunov function, more relaxed results are obtained. Different conditions in terms of linear matrix inequalities are provided. The proposed conditions are less demanding than the ones in the literature. This is illustrated via numerical examples.

A microsatellite genetic linkage map for Xiphophorus.

Interspecies hybrids between distinct species of the genus Xiphophorus are often used in varied research investigations to identify genomic regions associated with the inheritance of complex traits. There are 24 described Xiphophorus species and a greater number of pedigreed strains; thus, the number of potential interspecies hybrid cross combinations is quite large. Previously, select Xiphophorus experimental crosses have been shown to exhibit differing characteristics between parental species and among the hybrid fishes derived from crossing them, such as widely differing susceptibilities to chemical or physical agents. For instance, genomic regions harboring tumor suppressor and oncogenes have been identified via linkage association of these loci with a small set of established genetic markers. The power of this experimental strategy is related to the number of genetic markers available in the Xiphophorus interspecies cross of interest. Thus, we have undertaken the task of expanding the suite of easily scored markers by characterization of Xiphophorus microsatellite sequences. Using a cross between Xiphophorus maculatus and X. andersi, we report a linkage map predominantly composed of microsatellite markers. All 24 acrocentric chromosome sets of Xiphophorus are represented in the assembled linkage map with an average intergenomic distance of 7.5 cM. Since both male and female F1 hybrids were used to produce backcross progeny, these recombination rates were compared between "male" and "female" maps. Although several genomic regions exhibit differences in map length, male- and female-derived maps are similar. Thus Xiphophorus, in contrast to zebrafish, Danio rerio, and several other vertebrate species, does not show sex-specific differences in recombination. The microsatellite markers we report can be easily adapted to any Xiphophorus interspecies and some intraspecies crosses, and thus provide a means to directly compare results derived from independent experiments.

A point mutation in the 14alpha-sterol demethylase gene cyp51A contributes to itraconazole resistance in Aspergillus fumigatus.

The genes encoding 14alpha-sterol demethylases (cyp51A and cyp51B) were analyzed in 12 itraconazole (ITC)-resistant and three ITC-susceptible clinical isolates of Aspergillus fumigatus. Six ITC-resistant strains exhibited a substitution of another amino acid for glycine at position 54, which is located at a very conserved region of the Cyp51A protein. The cyp51A gene from the A. fumigatus wild-type strain (CM-237) was replaced with the mutated cyp51A gene copy of an ITC-resistant strain (AF-72). Two transformants exhibited resistance to ITC, both of which had incorporated the mutated copy of the cyp51A gene.

Flucytosine primary resistance in Candida species and Cryptococcus neoformans.

The in vitro activity of flucytosine (5FC) against 1,140 clinical isolates of Candida spp. and Cryptococcus neoformans was evaluated and compared with the activity of amphotericin B, fluconazole and itraconazole. Overall, 87.72% (1,000/1,140) of yeasts were susceptible to 5FC. This agent showed less potent in vitro activity against Candida glabrata, Candida krusei, Candida guilliermondii and Cryptococcus neoformans (MIC90s, 8-16 microg/ml) and intermediate activity or resistance to 6.5% of Candida albicans, 5.1% of Candida tropicalis and 0.8% of Candida parapsilosis strains. Amphotericin B showed potent activity against isolates with an MIC of 5FC > or = 8 microg/ml. A total of 112 of 140 strains that were SFC-intermediate or -resistant showed decreased susceptibility to azoles (P < 0.01).

Detection of resistance to amphotericin B in Candida isolates by using Iso-Sensitest broth.

A major limitation of the National Committee for Clinical Laboratory Standards M27-A methodology is reliable detection of amphotericin B (AMB) resistance. The results obtained by using Iso-Sensitest, a synthetic medium, to detect AMB resistance were analyzed and compared with those obtained with RPMI and antibiotic medium 3 (AM3). The ability to detect AMB resistance with RPMI is not enhanced by using a higher inoculum, glucose supplementation at a final concentration of 20 g/liter, spectrophotometric reading, or 24 h of incubation time. Testing using AM3 and an inoculum of 10(3) CFU/ml detects resistance. Identification of resistant isolates is not improved by glucose supplementation, changes in reading method, or changes in incubation time. However, the use of AM3 as assay medium and an inoculum of 10(5) CFU/ml did not allow detection of AMB resistance. Testing using Iso-Sensitest medium appears to be similar to AM3 in detecting resistance. The most pronounced discrimination is achieved by testing in Iso-Sensitest supplemented with glucose and spectrophotometric reading after 24 h of incubation. The reproducibility of MIC testing was greatest for Iso-Sensitest-based procedures. Use of Iso-Sensitest produces both highly reproducible MICs and reliable identification of AMB-resistant Candida isolates.

Identification of two different 14-alpha sterol demethylase-related genes (cyp51A and cyp51B) in Aspergillus fumigatus and other Aspergillus species.

Two cyp51-related genes (cyp51A and cyp51B) encoding 14-alpha sterol demethylase-like enzymes were identified in the opportunistic human pathogen Aspergillus fumigatus. PCR amplification using degenerate oligonucleotides based on conserved areas of cytochrome P450 demethylases of other filamentous fungi and yeasts allowed the cloning and sequencing of two different homologue genes in A. fumigatus. Southern analysis confirmed that both genes hybridized to distinct genomic loci and that both are represented as single copies in the genome. Comparison of the deduced Cyp51A and Cyp51B proteins with the CYP51 proteins from Penicillium italicum, Aspergillus nidulans, Erysiphe graminis, Uncinula necator, Botrytis cinerea, Ustilago maydis, Cryptococcus neoformans, Candida albicans, Saccharomyces cerevisiae, Candida tropicalis, and Candida glabrata showed that the percentages of identity of the amino acid sequences (range, 40 to 70%) were high enough to consider Cyp51A and Cyp51B to be members of the fungal CYP51 family. Fragments from both genes were also cloned from other Aspergillus spp. (A. flavus, A. nidulans, and A. terreus). Phylogenetic analysis showed that, at least in the most pathogenic species of Aspergillus, there are two fungal CYP51 proteins. This is the first report of the existence of two homologue genes coding for 14-alpha sterol demethylase in the fungal kingdom. This finding could provide insights into the azole resistance mechanisms operating in fungi. The primers used here may be useful molecular tools for facilitating the cloning of novel 14-alpha sterol demethylase genes in other filamentous fungi.

Standardization of antifungal susceptibility variables for a semiautomated methodology.

Recently, the methodology that will serve as a basis of the standard for antifungal susceptibility testing of fermentative yeasts of the European Committee on Antibiotic Susceptibility Testing has been described. This procedure employs a spectrophotometric method for both inoculum adjustment and endpoint determination. However, the utilization of a spectrophotometer requires studies for standardization. The present work analyzes the following parameters: (i) accuracy of inoculum preparation, (ii) correlation between optical density and CFU per milliliter, (iii) influence of the wavelength on the endpoint determination, and (iv) influence of the dimethyl sulfoxide concentration on the growth kinetics. The main results can be summarized as follows: (i) inoculum preparation following the methodology recommended by the National Committee for Clinical Laboratory Standards is an exact procedure; (ii) the relationship between optical density and CFU per milliliter is linear (coefficient of determination, r(2) = 0.84); (iii) MICs obtained by means of spectrophotometric readings at different wavelengths are identical (for amphotericin B, an intraclass correlation coefficient of 0.98 was obtained; for fluconazole, the intraclass correlation coefficient was 1); and (iv) a 2% concentration of dimethyl sulfoxide produces a significantly slower and lower growth curve of Candida spp. than other concentrations.

Azasordarins: susceptibility of fluconazole-susceptible and fluconazole-resistant clinical isolates of Candida spp. to GW 471558.

The in vitro activity of the azasordarin GW 471558 was compared with those of amphotericin B, flucytosine, itraconazole, and ketoconazole against 177 clinical isolates of Candida spp. GW 471558 showed potent activity against Candida albicans, Candida glabrata, and Candida tropicalis, even against isolates with decreased susceptibility to azoles. Candida krusei, Candida parapsilosis, Candida lusitaniae, and Candida guilliermondii are resistant to GW 471558 in vitro (MICs, >128 microg/ml).

Influence of glucose supplementation and inoculum size on growth kinetics and antifungal susceptibility testing of Candida spp.

The influences of inoculum size and glucose supplementation on the growth kinetics of 60 Candida spp. clinical isolates (Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata, Candida krusei, and Candida lusitaniae [10 isolates each]) are assessed. The combined influence of growth and reading method (visual or spectrophotometric) on the determination of the MICs of amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole, and voriconazole is also analyzed, and the MICs are compared with those determined by the National Committee for Clinical Laboratory Standards standard microdilution method (NCCLS document M27-A). Glucose supplementation and inoculum size had a significant influence on the growth cycles of these yeasts, and a statistically significant denser growth (optical density at 540 nm) was seen for both incubation periods, 24 and 48 h (P < 0.01). A longer exponential phase and shorter lag phase were also observed. The A540 values at 24 h of incubation with medium containing glucose and an inoculum of 10(5) CFU/ml were >0.4 U for all species, with the exception of that for C. parapsilosis (A540 = 0.26 +/- 0.025). The MICs at 24 h determined by testing with 2% glucose and an inoculum of 10(5) CFU/ml showed the strongest agreement (96.83%) with MICs determined by the reference method. MICs were not falsely elevated, and good correlation indexes were obtained. The reproducibility of results with this medium-inoculum combination was high (intraclass correlation coefficient, 0.955). The best agreement and reproducibility of results for spectrophotometric readings were achieved with endpoints of 50% growth inhibition for flucytosine and azoles and 95% for amphotericin B. Supplementation of test media with glucose and an inoculum size of 10(5) CFU/ml yielded a reproducible technique that shows elevated agreement with the reference procedures and a shorter incubation period for obtaining reliable MIC determinations. The spectrophotometric method offers an advantage over the visual method by providing a more objective and automated MIC determination.

Sustained gastrointestinal colonization and systemic dissemination by Candida albicans, Candida tropicalis and Candida parapsilosis in adult mice.

The ability of nine clinical isolates of Candida species (three C. albicans, three C. tropicalis and three C. parapsilosis) to colonize and invade the gastrointestinal (GI) tract of adult male CD-1 (ICR) mice was determined. The effect of dietary tetracycline plus glucose supplementation on colonization was evaluated. Strains were intragastrically inoculated. Tetracycline and glucose altered substantially aerobic flora, especially streptococci (average fall 1.1 +/-0.3 log(10) CFU/g, p<0.01 by the Student's t test). At two weeks after oral challenge, sustained and high colonization of GI tract by Candida (mean 5,28 +/- 0.18 log(10) CFU/g, p<0.01) was achieved in all mice receiving glucose-tetracycline supplementation, excepting in animals inoculated with one of C. tropicalis isolates. Histologic sections of the stomachs revealed multiple intraepithelial micro-abscesses associated with hyphae in the region of the cardial-atrium fold. Under immunosuppression, systemic spread of C. albicans and C. tropicalis was observed in 62% and 24% of animals receiving dietary supplementation respectively. Dissemination was not noted for C. parapsilosis isolates. We have developed a simple and inexpensive murine model of sustained colonization of GI tract. This model could be useful for analyzing prophylaxis, treatment and diagnosis of systemic Candida infections and for evaluating virulence of strains.

Susceptibility of fluconazole-resistant clinical isolates of Candida spp. to echinocandin LY303366, itraconazole and amphotericin B.

The in vitro activity of LY303366 was compared with those of itraconazole and amphotericin B against 156 fluconazole-resistant (MIC > or = 16 mg/L) clinical isolates of Candida spp. An adaptation of the NCCLS reference method was employed for determination of MICs. LY303366 was more potent than either itraconazole or amphotericin B against Candida albicans, Candida glabrata, Candida krusei and Candida tropicalis, even against isolates with itraconazole MICs > or = 1 mg/L. LY303366 was less potent in vitro against Candida parapsilosis and Candida guilliermondii isolates. LY303366 has promising antifungal activity and warrants further investigation.

Genetic similarity among one Aspergillus flavus strain isolated from a patient who underwent heart surgery and two environmental strains obtained from the operating room.

We report the simultaneous isolation of one Aspergillus flavus strain from the aortic prosthesis of a heart surgery patient and another two isolates recovered from a dual-reservoir cooler-heater used in the operating room where this patient was operated on. Genetic typing of these three isolates by randomly amplified polymorphic DNA (RAPD) revealed identical genotypes. Eight unrelated control strains of A. flavus had eight different genotypes. These results clearly indicated the nosocomial origin of the A. flavus strain isolated from the patient. We suggest that the RAPD technique is a rapid and reliable tool to ascertain the epidemiology of infections caused by A. flavus.

Characterization of a possible nosocomial aspergillosis outbreak.

OBJECTIVE: To study the epidemiologic aspects of a suspected outbreak of nosocomial invasive aspergillosis. METHODS: Sixteen Aspergillus fumigatus strains were isolated from bronchoalveolar washings or sputa of 10 patients during a 9-month period. Furthermore, two environmental samples, isolated in a microbiological screening of the hospital, were also available for analysis. Random amplified polymorphic DNA analysis (RAPD) was carried out. RESULTS: The analysis performed by RAPD clearly demonstrated substantial genetic variation among the isolates. Both of the two different primers selected for RAPD analysis (R-108 and AP12h) were able to demonstrate that the strains isolated from all patients infected with the same fungal species and the environmental samples were genotypically distinct. The results by RAPD typing demonstrated that this technique could detect variability among isolates of Aspergillus fumigatus from different patients and even from the same patient. CONCLUSIONS: RAPD genotyping proved that the outbreak of invasive aspergillosis consisted of a series of events, non-related, and probably not coming from the same source within the hospital. This type of analysis is an easy, quick and highly discriminatory technique that may help in planning epidemiologic studies of aspergillosis.

Molecular characterization by PCR-fingerprinting of Candida dubliniensis strains isolated from two HIV-positive patients in Spain.

Six Candida dubliniensis isolates were recovered from two HIV-infected individuals in the course of a prospective study of recurrent oral candidosis among HIV-positive patients in Spain. Candida albicans strains as well as non-albicans strains were also obtained from these two patients. C. dubliniensis strains were germ-tube-positive and produced abundant chlamydospores. Fingerprinting the genomic DNAs of these six C. dubliniensis with the C. albicans-specific probe 27A as well as karyotyping was performed to confirm the identification of these isolates. Further analysis of their genomic DNAs was performed by PCR-fingerprinting with the core sequence of phage M13, and they exhibited species-specific multilocus band patterns, clearly distinct from those of C. albicans isolates analyzed in this study and in a previous one (Diaz-Guerra 1997). Intraspecies variation was also seen among PCR patterns yielded by C. dubliniensis isolates from different patients. Although few strains have been analyzed, the use of this PCR-fingerprinting procedure is a promising tool for further epidemiologic studies with C. dubliniensis. The isolation of C. dubliniensis from Spanish HIV-infected patients contributes to the idea of widespread geographic distribution of this species.

Comparative in vitro activity of voriconazole and itraconazole against fluconazole-susceptible and fluconazole-resistant clinical isolates of Candida species from Spain.

The in vitro activity of voriconazole was compared with that of itraconazole against 299 fluconazole-susceptible (MIC < or = 8 microg/ml) and 130 fluconazole-resistant (MIC > or = 16 microg/ml) clinical isolates of Candida spp. An adaptation of the National Committee for Clinical Laboratory Standards reference method was employed for determination of MICs. Voriconazole showed more potent activity than either fluconazole and itraconazole, even against some Candida albicans, Candida glabrata, and Candida krusei isolates resistant to fluconazole. However, for fluconazole-resistant isolates, the MICs of itraconazole and voriconazole were proportionally higher than for fluconazole-susceptible isolates. These data may indicate cross-resistance.

[Development of a milk beverage based on pumpkin flakes]. / Desenvolvimento de bebida láctea a base de flocos de abóbora.

Vitamin A deficiency is one of the majors public health problems in Brazilian Northeast, and among other causes may be concerned to precocious weaning. Aiming at reducing this problem, a dehydrated product based on powdered milk and pumpkin flakes was developed to act as a carotene source at weaning period. Pumpkin flakes were obtained by drum drying at a 6 atm steam pressure, 0.75 m2 contact surface and 1 rpm, and had their content of carotenoids, beta carotene, centesimal composition molds and yeast and faecal coliforms evaluated. The flakes were added to sugar and whole powdered milk according to FAO/WHO nutritional recommendation to 6-12 months old children and submitted to acceptability test by a 6 judges' panel. The results showed that the drying process had a 7% efficiency score and the flakes composition presented 4.84% moisture; 4.0% protein; 5.5% ash; 1.30% fat; 6.22% fiber; 78.14% carbohydrates; 115.08 micrograms/g total carotenois and 80.64 micrograms/g beta-carotene content. No coliforms were detected and molds and yeast content was 4.0 x 10(2) CFU/g. The powdered formulation properly diluted in water supports 45% of the RDA for children (calories, protein, carbohydrates and lipids) and 100% vitamin A and protein considering a 400 ml/day ingestion.

Change in fluconazole susceptibility patterns and genetic relationship among oral Candida albicans isolates.

OBJECTIVE: To assess the genetic homogeneity or heterogeneity within each set of Candida albicans isolates colonizing/infecting the oral cavities of HIV-infected patients undergoing azole therapy when changes in susceptibility to fluconazole were detected. DESIGN: Fourteen HIV-positive patients suffering recurrent episodes of oral candidosis were prospectively followed from the first episode to the isolation of strains with decreased susceptibility to fluconazole. The strains of C. albicans isolated either from episodes or controls throughout the prospective study were analysed. METHODS: Electrophoretic karyotyping and hybridization with the repeated sequence probe 27A were used to delineate sequential isolates. In vitro susceptibility tests to fluconazole and ketoconazole were also performed. The results obtained by DNA fingerprinting with the probe combined with computer-assisted analysis were used to assess the genetic relationships amongst the strains. In addition, comparison with the genetic relatedness of a group of geographically unrelated strains was made. RESULTS: Isogenic populations of sequential isolates were observed only in two patients; 12 patients harboured heterogenic populations over time, although in 11 patients there was a predominant strain that was isolated more than once, and only one of these patients carried strains with a similarity index less than 80%. With the exception of two patients, each patient carried a major strain that became less susceptible to fluconazole. The similarity index for the unrelated strains was 59%. CONCLUSIONS: HIV-infected patients may carry a mixed population of strains, but the strains tend to be related to each other. The strains were maintained throughout the course of infection and at least one developed secondary resistance to fluconazole.

Comparison of four molecular typing methods for evaluating genetic diversity among Candida albicans isolates from human immunodeficiency virus-positive patients with oral candidiasis.

Candida albicans strain delineation by karyotyping. NotI restriction pattern analysis, hybridization with specific probe 27A, and PCR fingerprinting with the phage M13 core sequence were performed with 30 isolates from the oral cavities of 30 human immunodeficiency virus (HIV)-infected patients and 8 reference strains. Within the panel of clinical isolates, 20 were geographically related, although 10 isolates were susceptible to fluconazole and 10 isolates were resistant to fluconazole. The remaining isolates used in this study were fluconazole resistant and geographically unrelated. A composite DNA type was defined for each of the strains as the combination of types obtained by the four molecular methods. By this procedure, a great diversity of DNA types was found among isolates from the oropharynges of HIV-infected individuals with oral candidiasis. This diversity was not reduced when isolates were evaluated on the basis of whether they came from the same geographical locale and whether they were fluconazole resistant. These data refute the idea of a clonal origin for fluconazole-resistant strains among HIV-positive patients. Karyotyping was the least discriminatory method, yielding 19 DNA types among the 38 strains analyzed. Conversely, hybridization with the 27A probe showed a unique DNA pattern for each of the strains examined in this study. Our results demonstrate that at least two different molecular methods are needed for Candida albicans typing and that there is a great deal of strain variation within the species, irrespective of place of origin or antifungal resistance patterns.

Patterns of fluconazole susceptibility in isolates from human immunodeficiency virus-infected patients with oropharyngeal candidiasis due to Candida albicans.

We evaluated 119 episodes of oropharyngeal candidiasis due to C. albicans to study the patterns of fluconazole susceptibility of the isolates and the characteristics of the patients and to confirm the correlation between fluconazole susceptibility of isolates and therapeutic outcome. Sixty-one isolates were considered susceptible to fluconazole (MICs, < or = 0.5 microg/mL), 33 were intermediate (MICs, 1.0-8.0 microg/mL), and 25 were resistant (MICs, > or = 16.0 microg/mL). Patients infected with resistant strains had significantly lower CD4+ cell counts and a less recent AIDS diagnosis than patients infected with intermediate or susceptible strains. Previous fluconazole therapy and prophylaxis were significantly more frequent for patients infected with resistant and intermediate strains (P < .001). Decreased susceptibility to ketoconazole and itraconazole was observed in resistant and intermediate strains. Fluconazole treatment was ineffective for patients infected with resistant isolates; however, high doses of ketoconazole or itraconazole were successful for nine (81%) of them. Different patterns of fluconazole susceptibility among C. albicans strains are correlated with patients' characteristics and with therapeutic outcomes.