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

Erratum in: J Clin Microbiol 2001 Nov;39(11):4225.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.This work was supported in part by grant 1078/99 from Instituto de Salud Carlos III. T.M.D.-G. is a fellow of the Instituto de Salud Carlos III

Characterization of a possible nosocomial aspergillosis outbreak

ObjectiveTo study the epidemiologic aspects of a suspected outbreak of nosocomial invasive aspergillosis.MethodsSixteen 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.ResultsThe 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.ConclusionsRAPD 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

Plasmodium diversity in non-malaria individuals from the Bioko Island in Equatorial Guinea (West Central-Africa)

BACKGROUND: In this paper we analyse the Plasmodium sp. prevalence in three villages with different isolation status on the island of Bioko (Equatorial Guinea) where malaria is a hyper-endemic disease. We also describe the genetic diversity of P. falciparum, using several plasmodia proteins as markers which show a high degree of polymorphism (MSP-1 and MSP-2). The results obtained from three different populations are compared in order to establish the impact of human movements and interventions. METHODS: Plasmodium sp. were analysed in three villages on Bioko Island (Equatorial Guinea), one of which (Southern) is isolated by geographical barriers. The semi-nested multiplex polymerase chain reaction (PCR) technique was used to determine the prevalence of the four human plasmodia species. The genotyping and frequency of P. falciparum populations were determined by PCR assay target polymorphism regions of the merozoite surface proteins 1 and 2 genes (MSP-1 and MSP-2). RESULTS: The data obtained show that there are no differences in plasmodia population flow between the Northwest and Eastern regions as regards the prevalence of the different Plasmodium species. The Southern population, on the other hand, shows a minor presence of P. malariae and a higher prevalence of P. ovale, suggesting some kind of transmission isolated from the other two. The P. falciparum genotyping in the different regions points to a considerable allelic diversity in the parasite population on Bioko Island, although this is somewhat higher in the Southern region than the others. There was a correlation between parasitaemia levels and the age of the individual with the multiplicity of infection (MOI). CONCLUSION: Results could be explained by the selection of particular MSP alleles. This would tend to limit diversity in the parasite population and leading up to the extinction of rare alleles. On the other hand, the parasite population in the isolated village has less outside influence and the diversity of P. falciparum is maintained higher. The knowledge of parasite populations and their relationships is necessary to study their implications for control intervention

Susceptibility patterns and molecular identification of Trichosporon species

The physiological patterns, the sequence polymorphisms of the internal transcriber spacer (ITS), and intergenic spacer regions (IGS) of the rRNA genes and the antifungal susceptibility profile were evaluated for their ability to identify Trichosporon spp. and their specificity for the identification of 49 clinical isolates of Trichosporon spp. Morphological and biochemical methodologies were unable to differentiate among the Trichosporon species. ITS sequencing was also unable to differentiate several species. However, IGS1 sequencing unambiguously identified all Trichosporon isolates. Following the results of DNA-based identification, Trichosporon asahii was the species most frequently isolated from deep sites (15 of 25 strains; 60%). In the main, other Trichosporon species were recovered from cutaneous samples. The majority of T. asahii, T. faecale, and T. coremiiforme clinical isolates exhibited resistance in vitro to amphotericin B, with geometric mean (GM) MICs >4 mug/ml. The other species of Trichosporon did not show high MICs of amphotericin B, and GM MICs were <1 mug/ml. Azole agents were active in vitro against the majority of clinical strains. The most potent compound in vitro was voriconazole, with a GM MIC </=0.14 mug/ml. The sequencing of IGS correctly identified Trichosporon isolates; however, this technique is not available in many clinical laboratories, and strains should be dispatched to reference centers where these complex methods are available. Therefore, it seems to be more practical to perform antifungal susceptibility testing of all isolates belonging to Trichosporon spp., since correct identification could take several weeks, delaying the indication of an antifungal agent which exhibits activity against the infectious strain.S

Multi-band high resolution spectroscopy rules out the hot Jupiter BD+20 1790b - First data from the GIARPS Commissioning

Context. Stellar activity is currently challenging the detection of young planets via the radial velocity (RV) technique. Aims. We attempt to definitively discriminate the nature of the RV variations for the young active K5 star BD+20 1790, for which visible (VIS) RV measurements show divergent results on the existence of a substellar companion. Methods. We compare VIS data with high precision RVs in the near infrared (NIR) range by using the GIANO - B and IGRINS spectrographs. In addition, we present for the first time simultaneous VIS-NIR observations obtained with GIARPS (GIANO - B and HARPS - N) at Telescopio Nazionale Galileo (TNG). Orbital RVs are achromatic, so the RV amplitude does not change at different wavelengths, while stellar activity induces wavelength-dependent RV variations, which are significantly reduced in the NIR range with respect to the VIS. Results. The NIR radial velocity measurements from GIANO - B and IGRINS show an average amplitude of about one quarter with respect to previously published VIS data, as expected when the RV jitter is due to stellar activity. Coeval multi-band photometry surprisingly shows larger amplitudes in the NIR range, explainable with a mixture of cool and hot spots in the same active region. Conclusions. In this work, the claimed massive planet around BD+20 1790 is ruled out by our data. We exploited the crucial role of multi- wavelength spectroscopy when observing young active stars: thanks to facilities like GIARPS that provide simultaneous observations, this method can reach its maximum potential.Comment: 12 pages, 7 figure

Évaluation de la quantité et la qualité des eaux de ruissellement pour les systèmes modulaires de toiture végétalisée extensive

Colloque avec actes et comité de lecture. Internationale.International audienc

The effect of intra-articular injection of autologous bone marrow stem cells on pain and knee function in patients with osteoarthritis

Abstract Aim: Management of osteoarthritis (OA) is basically symptomatic. Recently, stem cells (SC) have been used in the search for an optimum treatment. We decided to conduct a controlled clinical trial to determine if a single intra-articular injection of in vivo stimulated bone marrow SC could lead to an improvement in pain management and quality of life in patients with knee OA. Method: This was a prospective, open-label, phase I/II clinical trial to assess the safety and efficacy of a single intra-articular injection of autologous stimulated bone marrow stem cells (BM-SC) in patients with knee OA. Individuals of both genders older than 30 years with confirmed diagnosis of OA who signed informed consent were included in two groups: SC group received in vivo BM stimulation with subcutaneous administration of granulocyte colony stimulating factor (G-CSF). SC were obtained by BM aspiration and administered in a single intra-articular injection. The control group received exclusively oral acetaminophen. Visual analogue scale and Western Ontario and McMaster Universities Osteoarthritis Index scores were performed at 1 week, 1 month and 6 months in both groups. This trial was registered in ClinialTrials.gov NCT01485198. Results: A total of 61 patients were included. Socio-demographic characteristics, OA grades and initial scores were similar in both groups. The BM-SC group showed significant improvement in knee pain and quality of life during the 6-month follow-up. Conclusion: The study demonstrates feasibility and supports efficacy of a completely ambulatory procedure in treatment of knee OA. Key words: bone marrow, knee osteoarthritis, stem cells