Typing of Saccharomyces cerevisiae clinical strains by using microsatellite sequence polymorphism.

It seems that S. cerevisiae, which was thought for about 30 years to be a nonpathogenic yeast, should now be considered an opportunistic pathogen. In this study, we estimated the discrimination ability of the microsatellite sequence amplification technique within a sample of clinical and reference S. cerevisiae strains and S. boulardii reference strains.

In vitro susceptibility testing of Candida and Aspergillus spp. to voriconazole and other antifungal agents using Etest: results of a French multicentre study.

Minimum inhibitory concentrations (MICs) of the antifungal agent voriconazole were determined using the Etest and compared with those of amphotericin B, itraconazole and fluconazole using 1986 clinical isolates of Candida spp. Voriconazole MICs were also compared with those of amphotericin B and itraconazole using 391 clinical isolates of Aspergillus spp. Voriconazole was found to have more potent activity and lower MIC values than amphotericin B, itraconazole and fluconazole against C. albicans, C. tropicalis, C. parapsilosis and C. kefyr. Against C. glabrata and C. krusei, voriconazole was more active than either of the other two azole antifungals but had similar activity to amphotericin B. For species of Aspergillus, MIC values of voriconazole were lower than those of amphotericin B and itraconazole against A. fumigatus and A. flavus, and were similar to those of amphotericin B against A. niger. Against A. terreus, MIC values for voriconazole and itraconazole were similar. A. terreus is known to be resistant to amphotericin B, and this was reflected in higher MIC values compared with those of voriconazole and itraconazole. Voriconazole therefore compares very favourably with other antifungal agents against a large number of clinical isolates of Candida and Aspergillus spp.

Effect of medium composition on static and cidal activity of amphotericin B, itraconazole, voriconazole, posaconazole and terbinafine against Aspergillus fumigatus: a multicenter study.

The effect of the medium composition on the fungistatic (MIC) and fungicidal (MLC) activity of amphotericin B, itraconazole, voriconazole, posaconazole and terbinafine against four Aspergillus fumigatus strains has been investigated by four European laboratories. MICs were determined by broth microdilution, using RPMI 1640 and Antibiotic Medium 3 (AM3), three times in three independent determinations by the four laboratories. MLCs were determined for the three independent determinations by the four laboratories, subculturing 100 microl from each well showing no visible growth after 48 hours. Except for a 2-dilution difference observed in three cases, no differences were observed between MICs determined on the two media. In contrast, a 3- to 6-dilution discrepancy between the MLCs was observed for the azoles. Endpoints on RPMI were higher than those on AM3. A 1-2 dilution difference was noted between both the endpoints of amphotericin B and of terbinafine. The highest inter- and intra-laboratory agreements were reached on AM3. The azoles showed a medium-dependent fungicidal activity.

Clonality structure in Candida dubliniensis.

Multilocus enzyme electrophoresis was performed on 76 European strains of Candida dubliniensis. Ten of the 20 enzyme-encoding loci were polymorphic, giving rise to 10 electrophoretic types within the sample studied. Investigation of the population genetics of a subset of 36 strains from HIV-infected patients in London showed the existence of strong heterozygote deficits and excesses associated with significant linkage disequilibria between pairs of loci. These findings, together with the predominance of multilocus genotypes, strongly suggest that C. dubliniensis is mainly (if not totally) clonal. Analysis of genotypes of a larger number of strains should confirm this conclusion and improve our understanding of the epidemiology of this pathogen.

Genetic structure of Candida glabrata populations in AIDS and non-AIDS patients.

The genotypes of 63 strains (11 reference strains and 52 strains from hospitalized patients) of the haploid yeast Candida glabrata were determined from 33 putative gene enzymatic loci. This enabled the characterization of 26 different multilocus genotypes. Genetic differentiation was found between distant hospitals (located in Montpellier and Paris, France) but not for other parameters (anatomic origins or human immunodeficiency virus-positive [HIV+] and HIV- patients). Strong nonrandom association between loci could be seen. Such statistical linkages were confirmed upon comparing the patterns of 14 RAPD [random(ly) amplified polymorphic DNA] primers from 20 of these strains to results obtained from multilocus enzyme electrophoresis analysis. This finding suggests a mainly clonal mode of reproduction of C. glabrata. The consequences of the clonality displayed by C. glabrata populations on the epidemiology of this yeast are also discussed.

Synthesis and characterization of the antitumor activities of analogues of meridine, a marine pyridoacridine alkaloid.

Marine compounds with pyridoacridine skeletons are known to exhibit interesting antitumor activities. Among these compounds, meridine has already been reported as having significant antitumor activities in vitro. We synthesized 24 analogues of meridine substituted on ring A with the aim of obtaining compounds that display significantly higher in vitro antitumor activities than meridine. The 24 compounds and meridine used as a control compound were tested at 6 different concentrations on 12 different human cancer cell lines including various histopathological types (glioblastomas and breast, colon, lung, prostate, and bladder cancers). The IC(50) value (i.e., the drug concentration inhibiting the mean growth value of the 12 cell lines by 50%) of these 25 compounds ranged over 5 log concentrations, i.e., between 10 and 0.0001 microM, with four of the compounds exhibiting a significantly higher in vitro antitumor activity than meridine. These compounds will now be subjected to further pharmacological investigation including in vivo testing on both conventional murine tumors and human tumors grafted onto nude mice.

Genetic polymorphism of Aspergillus fumigatus in clinical samples from patients with invasive aspergillosis: investigation using multiple typing methods.

The genotypes of 52 strains of Aspergillus fumigatus isolated from 12 patients with invasive aspergillosis were investigated using three typing methods (random amplified polymorphic DNA, sequence-specific DNA polymorphism, and microsatellite polymorphism) combined with multilocus enzyme electrophoresis. Isolates were from patients hospitalized in three different geographic areas (Lyon, France; Grenoble, France; and Milan, Italy). In each case, the genetic polymorphism of several colonies (two to five) within the first respiratory clinical sample was studied. For the 52 isolates tested, random amplified polymorphic DNA identified 8 different genotypes, sequence-specific DNA polymorphism identified 9 different types, and microsatellite polymorphism identified 14 types. A combination of these results with multilocus enzyme electrophoresis study identified 25 different types within the sample studied. We identified 3 patients (of the 12 studied) who carried a single genotype; 6 patients were infected by two genotypes, 1 patient had four genotypes, while the last patient had five. A combination of typing methods provided better discrimination than the use of a single method. Typing methods revealed a population structure within each geographical site, suggesting that the epidemiology of A. fumigatus should be considered separately for each of these geographic areas. This study demonstrates the usefulness of combining several typing methods in reaching an understanding of the epidemiology of A. fumigatus and clarifies whether it is sufficient to type one isolate from each specimen to determine the strain involved in invasive aspergillosis.

Rapid therapeutic response onset of a new pharmaceutical form of chloroquine phosphate 300 mg: effervescent tablets.

OBJECTIVE: To compare the efficiency, safety and taste of two pharmaceutical forms of chloroquine phosphate 300 mg: effervescent tablets against uncoated tablets. METHOD: An open randomized study with 60 adults who suffered from acute uncomplicated Plasmodium falciparum malaria in three health centres in Nkongsamba health district, Cameroon. RESULTS: Mean times to fever clearance, symptoms clearance and asexual parasites clearance were longer in the uncoated tablets group: 36 h (range 24-48 h, SD = 16.8) vs. 60 h (range 24-96 h, SD = 31.2, P = 0.001) for fever clearance, 36 h (24-48 h, SD = 16.8) vs. 48 h (24-72, SD = 24, P = 0.001) for symptoms clearance and 48 h (24-72, SD = 1) vs. 72 h (48-96, SD = 24, P = 0.001) for parasitaemia clearance. Uncoated tablets took significantly longer to achieve 50% reduction of the initial asexual parasite density: (mean/SD) 19.2 h/7 vs. 52.8 h/16.8, P < 0.00001. The adverse effects in the two groups were similar, P > 0.05. The cure rate at day 7 in the two groups was similar, P > 0.05. There was no chloroquine resistance in the effervescent tablets group but one RI and one RII resistance in the uncoated tablets group. The taste of the two pharmaceutical forms was significantly different, P < 0.00001. Effervescent tablets tasted sweet (score = 7.93), whereas uncoated tablets were bitter (score = 2.07). CONCLUSION: Effervescent tablets of chloroquine phosphate 300 mg work faster than uncoated tablets and because of their safe use and sweet taste achieve good therapeutic compliance.

Genotypic identification of Candida dubliniensis isolated from HIV patients by MLEE.

Candida dubliniensis is a novel species only recently described. This emerging pathogen shares some of the phenotypic characteristics specific to C. albicans but is genetically different. In this study we typed four strains of atypical C. albicans isolated in our laboratory and compared them to 41 strains of C. albicans and 11 strains of C. dubliniensis by several phenotypic methods and by multilocus enzyme electrophoresis. Using factorial correspondence analysis, we distinguished C. dubliniensis and the atypical C. albicans strains from all strains of C. albicans. Atypical C. albicans strains were identified as C. dubliniensis.

The first synthesis of the pentacyclic pyridoacridine marine alkaloids: arnoamines A and B.

The synthesis of the marine pyridoacridine alkaloids arnoamines A and B has been accomplished in six and seven steps from 4-chloro-8-methoxy-5-nitroquinoline in 13% and 4% overall yield, respectively.

Triflusulfuron-methyl dissipation in water and soil.

This paper reports laboratory studies of the behavior and fate of triflusulfuron-methyl in aqueous buffer and soils. Aqueous hydrolysis was pH-dependent and fast in acidic buffer solutions. In basic buffers, the hydrolysis rate variation was low between pH 7 and pH 10. The degradation pathway in the range of pH 4-10 was via cleavage of the sulfonylurea bridge to form two transformation products: 2-amino-4-(dimethylamino)-6-(2,2,2-trifluoroethoxy)-1,3, 5-triazine (2) and 6-methyl-2-methylcarboxylate benzene sulfonamide (3). Comparison of transformation rates in sterile and nonsterile soils indicates that chemical and microbial processes are important in soil degradation. The former is more important in acidic soils, and the latter is more important in basic soils. A biphasic model fits well with dissipation of triflusulfuron-methyl in soil. The triazine formed during the first step of transformation was degraded more rapidly in basic soils than in acidic soils.

Multicentric genetic study of Candida albicans isolates from non-neutropenic patients using MLEE typing: population structure and mode of reproduction.

A mycological survey was conducted on non-neutropenic patients in three distinct intensive care units in two hospitals in Marseille (France) from November 1993 to November 1995. Candida albicans positive cultures from 62 patients were included in this study. Every first isolate of each patient was typed by multilocus enzyme electrophoresis (MLEE). The enzyme profiles obtained from 15 polymorphic loci were then compared. This analysis demonstrated a strong population differentiation of C. albicans infective strains within and between the different care units and confirmed the probable preponderant clonal mode of reproduction of this yeast.

Case report of three Candida albicans infections detected at delivery.

We report three similar cases of Candida albicans infections in neonates, at delivery. A retrospective study of the isolates was conducted to define the diversity of infective strains and their susceptibility to amphotericin B and fluconazole. Three neonates with fever, 'not doing well' at delivery had positive cultures for C. albicans. Samples were then taken from the mothers who did not exhibit any clinical symptoms of infection. Candida albicans strains isolated from both neonates and mothers were cultured, six colonies of each were typed by multilocus enzyme electrophoresis. The E-test method was used to determine the susceptibility of each colony to the two antifungals commonly used in this unit: amphotericin B and fluconazole. The initial isolates were composed of different types of strains. In the three cases, one of the mother types was found in the neonate isolates, leading us to suggest a vertical transmission of strains. All of the other types were distinct. All of the types were susceptible to amphotericin B, although three of them, one type isolated from a neonate and two types isolated from the mother, were resistant to fluconazole. The diversity of infective strains remains alarming and encourages the consideration of several colonies per isolate or several isolates, when it is possible, per infection case. This study also points out the need to survey the susceptibility of infective strains, since some of them appear soon to be resistant to fluconazole.

Multilocus enzyme electrophoresis analysis of Aspergillus fumigatus strains isolated from the first clinical sample from patients with invasive aspergillosis. EBGA Network. European Research Group on Biotype and Genotype of Aspergillus.

The genotypes of 50 isolates of Aspergillus fumigatus from 11 patients with invasive aspergillosis, obtained from three hospitals in different geographical areas, were determined by multilocus enzyme electrophoresis (MLEE). The study analysed the genetic polymorphism of multiple isolates from the first sample. Seven of the 14 enzymic loci studied were polymorphic, giving rise to eight different electrophoretic types. For nine of 11 patients studied, no polymorphism was observed in isolates within the first clinical sample. Analysis of genetic distance between electrophoretic types demonstrated a genetic heterogeneity within each geographical site. Moreover, some genotypes were preferentially found in a given area and this revealed a population structure within these geographical sites. Therefore, the epidemiology of A. fumigatus should be considered separately for each of these areas. The multiple discriminatory markers of MLEE seem to provide a powerful tool for increasing the understanding of the biology of this fungus.

Genetic diversity in the yeast species Malassezia pachydermatis analysed by multilocus enzyme electrophoresis.

Fifty-two strains of the yeast species Malassezia pachydermatis were analysed by multilocus enzyme electrophoresis. M. pachydermatis appeared to be genetically heterogeneous. A total of 27 electrophoretic types were identified that could be divided into five distinct groups with different host specificities. The diversity revealed by this electrophoretic method matched remarkably well the reported genetic variability obtained by comparing large subunit rRNA sequences. This study also suggests that genetic exchanges can occur in the anamorphic species M. pachydermatis.

[Genotype, serotype and sensitivity to fluconazole of Candida albicans strains isolated from HIV-positive patients]. / Génotype, sérotype et sensibilité au fluconazole de souches de Candida albicans isolées chez des patients VIH positif.

Genotype, serotype and susceptibility in vitro to fluconazole of 104 C. albicans strains isolated from HIV+ patients were studied. The possible correlations between genotype analysed by multilocus enzyme electrophoresis (MLEE) and phenotype of medical relevance (serotype and susceptibility to fluconazole) of Candida albicans isolated from these patients treated with fluconazole were evaluated by factorial correspondence analysis. No correlation was observed between genotype and in vitro or clinical response to fluconazole. In counterpart, serotype B C. albicans was associated with some multilocus genotypic patterns.

In vitro antimalarial activity and cytotoxicity of cochlospermum tinctorium and C. planchonii leaf extracts and essential oils.

The antimalarial and toxicological properties of Cochlospermum tinctorium and C. planchonii extracts and essential oils prepared from their leaves were studied. The oil components were extracted by hydrodistillation of the plant leaves and characterized by gas chromatography and mass spectrometry. Crude extracts and oils were tested for in vitro antimalarial activity on Plasmodium falciparum. The IC50 were evaluated after 24 and 72 h contact between the oils and the parasite culture, and ranged from 22 to 500 micrograms/ml. C. planchonii leaf oil yielded the best antimalarial effect (IC50: 22-35 micrograms/ml), while the most potent effect from crude leaf extracts was induced by C. tinctorium. The cytotoxicity of the leaf crude extracts and oils was assessed on the K562 cell line and showed IC50 values ranging between 33 and 2000 micrograms/ml.

Genetic multilocus studies of different strains of Cryptococcus neoformans: taxonomy and genetic structure.

The genotypes of 107 strains of Cryptococcus isolated from the environment or from patients from various geographical areas were determined by multilocus enzyme electrophoresis (MLEE). We analyzed the relationships between genotype structure and serotype and between genotype structure and strain origin. Twelve of the 14 enzyme-encoding loci studied were polymorphic, giving rise to 48 electrophoretic types. The genotypes of C. neoformans and C. laurentii were very similar. MLEE could not distinguish between these two pathogenic species. A correlation between the genetic multilocus structure and the origin of the sample (from the environment or patients) existed. A second analysis detected a correlation between genotype distribution and serotype. The second analysis considered three serotype groups (B, C, and A plus D plus A/D), proving that serotypes A, D, and A/D are closely related. MLEE is a useful epidemiological tool for improving our understanding of the biology of this fungus.