Alexis Valentin

Routine Identification of Medical Fungi by the New Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight System with a New Time-Effective Strategy

ABSTRACT We report here a clinical evaluation of the Vitek MS system for rapid fungal identification. A strategy that uses a single deposit without prior protein extraction was utilized to save time and money. Clinical isolates from the Toulouse University hospital were used to evaluate the performance of the Vitek MS compared to that of both routine laboratory techniques and Vitek2. The Vitek MS performed well in the identification of yeasts and Aspergillus fungi (93.2% of correct identifications).

Oregano: Chemical Analysis and Evaluation of Its Antimalarial, Antioxidant, and Cytotoxic Activities

GC-FID and GC-MS analysis of essential oil from oregano leaves (Origanum compactum) resulted in the identification of 46 compounds, representing more than 98% of the total composition. Carvacrol was the predominant compound (36.46%), followed by thymol (29.74%) and p-cymene (24.31%). Serial extractions with petroleum ether, ethyl acetate, ethanol, and water were performed on aerials parts of Origanum compactum. In these extracts, different chemical families were characterized: polyphenols (gallic acid equivalent 21.2 to 858.3 g/kg), tannins (catechin equivalent 12.4 to 510.3 g/kg), anthocyanins (cyanidin equivalent 0.38 to 5.63 mg/kg), and flavonoids (quercetin equivalent 14.5 to 54.7 g/kg). The samples (essential oil and extracts) were subjected to a screening for antioxidant (DPPH and ABTS assays) and antimalarial activities and against human breast cancer cells. The essential oil showed a higher antioxidant activity with an IC50=2±0.1 mg/L. Among the extracts, the aqueous extract had the highest antioxidant activity with an IC50=4.8±0.2 mg/L (DPPH assay). Concerning antimalarial activity, Origanum compactum essential oil and ethyl acetate extract showed the best results with an IC50 of 34 and 33 mg/mL, respectively. In addition, ethyl acetate extract (30 mg/L) and ethanol extract (56 mg/L) showed activity against human breast cancer cells (MCF7). The oregano essential oil was considered to be nontoxic.

Schistosomiasis Haematobium, Corsica, France

To the Editor: In Europe, urinary schistosomiasis (1) has previously been detected only in Portugal, where this focus disappeared during the 1950s (2). However, freshwater snails of the species Bulinus contortus, B. truncatus, and Planorbarius metidjensis, which are recognized intermediate hosts for Schistosoma haematobium trematodes, have been found in Portugal (3), Spain (4), and Corsica (5,6). This finding suggested that autochthonous schistosomiasis could re-emerge in southern Europe if these mollusks become infected. We report a probable focus for transmission of schistosomiasis haematobium in Corsica, France. In March 2014, a 4-year-old girl (index case-patient) from France was referred to the Toulouse University Hospital (Toulouse, France), with gross hematuria. Ultrasonography and cystoscopic examination of the bladder detected a polyp. Examination of the polyp for parasites identified bodies that were consistent with schistosome eggs. Parasitologic examination of urine confirmed schistosomiasis by detecting viable S. haematobium eggs. The parents of the girl (family A) did not report any stay or travel in an area to which urinary schistosomiasis was endemic; they reported summer holidays only in Mallorca in the Balearic Islands (Spain) and Corsica. However, her father reported that since 2012, he had experienced gross hematuria that had been evaluated by standard urologic investigations but not by cystoscopy; no etiology was determined. Parasitologic urinalysis in our hospital department showed numerous viable S. haematobium eggs in the father’s urine. The parents of the index case-patient also reported that an 8-year-old boy in a friend’s family (family B), who shared summer vacations with them had exhibited gross hematuria since February 2013. A third family (family C) was also investigated because they also spent holidays in Corsica with families A and B. Families B and C had also spent a summer in Mallorca, but they denied any contact with freshwater. Of 11 French native-born members of the 3 families, 6 had positive results for S. haematobium by urine examination. All case-patients had specific positive immunodiagnostic results by an ELISA that used S. mansoni extracts and by indirect hemagglutination. In addition, 2 family members who had a negative result by urine examination had a positive serologic result. Spending summer vacations in the same village in Corsica (Sainte-Lucie de Porto-Vecchio), where members of the 3 families had bathed at least once per holiday period in the Cavu River, was the epidemiologically prominent feature that linked these persons. Families A and C were in Sainte-Lucie de Porto-Vecchio in August 2011, and families A, B, and C were in the same location in August 2013. During these investigations, we were contacted by the Department of Tropical Medicine, Dusseldorf University Hospital (Dusseldorf, Germany), because a 10-year-old boy and his father had been given diagnoses of schistosomiasis haematobium on the basis of positive urinalysis results for S. haematobium eggs. Two other members of this family (5 persons) had a positive immunodiagnostic result. Locations of previous vacations for this family outside Germany included Spain (not the Balearic Islands) and Corsica, where they bathed frequently in the Cavu River. These epidemiologic findings provide strong circumstantial evidence supporting the presence of a previously unrecognized focus of urinary schistosomiasis in Corsica. We performed molecular analysis of schistosomal miracidia DNA. The second internal transcribed spacer region of the ribosomal gene complex (7,8) was amplified and sequenced. Viable eggs obtained from the patients in France were those of S. haematobium. Additional molecular investigations are being conducted to assess genetic diversity of this isolate from Corsica and the geographic origin of the introduced parasite. The malacologic situation in Sainte Lucie de Porto-Vecchio was investigated during May 12–19, 2014; three rivers (Figure) were included in the survey. Four sites were sampled in the Cavu River, and B. truncatus snails were found in 3 sites that corresponded to bathing areas (site 1: 41°43′53.57″N, 9°17′36.70″E; site 2: 41°43′22.13″N, 9°17′59.87″E; site 3: 41°42′8.40″N, 9°21′5.82″E). Snails were also found in the nearby Tarcu River (site 5) and Osu River (site 6). These findings confirmed previous data for the presence of B. truncatus snails in Corsica (5,6). Water temperature was recorded at 11:00 am at these 3 sites (range 15°C–16°C). This temperature range is not optimal for the snail intermediate host stage of the parasite life cycle (9,10). Of 148 live snails that were obtained in the Cavu River, none were infected with schistosome cercariae. Figure Corsica, France, showing malacologic survey sampling sites (oval) in 3 rivers (Tarcu, Cavu, and Osu). Bulinus truncatus snails were found at sites 1, 2, 3, 5, and 6. Data from the field survey and epidemiologic information for the cases in France and Germany, indicated transmission of schistosomiasis haematobium in the Cavu River in southeastern Corsica in 2011 and 2013. Additional supportive evidence is the fact that the father of the index case-patient had gross hematuria in 2012 and 2013. Two hypotheses are proposed to account for this situation. The first hypothesis is that the parasite (i.e., schistosome eggs) was transmitted by an infected person into the Cavu River in June or July 2011, when environmental conditions were favorable for snail infection. However, questions arise about survival of infected snails during the winter and their ability to reinfect the area during the following summers in 2012 and 2013. The second hypothesis is that schistosome eggs were spread by infected persons at the beginning of summer and caused a permanent transmission cycle in this focus. This situation would be difficult to control. Additional information should be obtained by a long-term malacologic survey to detect infected mollusks in this region.

PPARγ Ligands Switched High Fat Diet-Induced Macrophage M2b Polarization toward M2a Thereby Improving Intestinal Candida Elimination

Obesity is associated with a chronic low-grade inflammation that predisposes to insulin resistance and the development of type 2 diabetes. In this metabolic context, gastrointestinal (GI) candidiasis is common. We recently demonstrated that the PPARγ ligand rosiglitazone promotes the clearance of Candida albicans through the activation of alternative M2 macrophage polarization. Here, we evaluated the impact of high fat diet (HFD)-induced obesity and the effect of rosiglitazone (PPARγ ligand) or WY14643 (PPARα ligand) both on the phenotypic M1/M2 polarization of peritoneal and cecal tissue macrophages and on the outcome of GI candidiasis. We demonstrated that the peritoneal macrophages and the cell types present in the cecal tissue from HF fed mice present a M2b polarization (TNF-αhigh, IL-10high, MR, Dectin-1). Interestingly, rosiglitazone induces a phenotypic M2b-to-M2a (TNF-αlow, IL-10low, MRhigh, Dectin-1high) switch of peritoneal macrophages and of the cells present in the cecal tissue. The incapacity of WY14643 to switch this polarization toward M2a state, strongly suggests the specific involvement of PPARγ in this mechanism. We showed that in insulin resistant mice, M2b polarization of macrophages present on the site of infection is associated with an increased susceptibility to GI candidiasis, whereas M2a polarization after rosiglitazone treatment favours the GI fungal elimination independently of reduced blood glucose. In conclusion, our data demonstrate a dual benefit of PPARγ ligands because they promote mucosal defence mechanisms against GI candidiasis through M2a macrophage polarization while regulating blood glucose level.

Chemical composition and anticancer, antiinflammatory, antioxidant and antimalarial activities of leaves essential oil of Cedrelopsis grevei

The essential oil from Cedrelopsis grevei leaves, an aromatic and medicinal plant from Madagascar, is widely used in folk medicine. Essential oil was characterized by GC-MS and quantified by GC-FID. Sixty-four components were identified. The major constituents were: (E)-β-farnesene (27.61%), δ-cadinene (14.48%), α-copaene (7.65%) and β-elemene (6.96%). The essential oil contained a complex mixture consisting mainly sesquiterpene hydrocarbons (83.42%) and generally sesquiterpenes (98.91%). The essential oil was tested cytotoxic (on human breast cancer cells MCF-7), antimalarial (Plasmodium falciparum), antiinflammatory and antioxidant (ABTS and DPPH assays) activities. C. grevei essential oil was active against MCF-7 cell lines (IC50=21.5 mg/L), against P. falciparum, (IC50=17.5mg/L) and antiinflammatory (IC50=21.33 mg/L). The essential oil exhibited poor antioxidant activity against DPPH (IC50>1000 mg/L) and ABTS (IC50=110 mg/L) assays. A bibliographical review was carried out of all essential oils identified and tested with respect to antiplasmodial, anticancer and antiinflammatory activities. The aim was to establish correlations between the identified compounds and their biological activities (antiplasmodial, anticancer and antiinflammatory). According to the obtained correlations, 1,4-cadinadiene (R(2)=0.61) presented a higher relationship with antimalarial activity. However, only (Z)-β-farnesene (R(2)=0.73) showed a significant correlation for anticancer activity.

Flow cytometry for the evaluation of anti-plasmodial activity of drugs on Plasmodium falciparum gametocytes.

BackgroundThe activity of promising anti-malarial drugs against Plasmodium gametocytes is hard to evaluate even in vitro. This is because visual examination of stained smears, which is commonly used, is not totally convenient. In the current study, flow cytometry has been used to study the effect of established anti-malarial drugs against sexual stages obtained from W2 strain of Plasmodium falciparum. Gametocytes were treated for 48 h with different drug concentrations and the gametocytaemia was then determined by flow cytometry and compared with visual estimation by microscopy.Results and conclusionsInitially gametocytaemia was evaluated either using light microscopy or flow cytometry. A direct correlation (r2 = 0.9986) was obtained. Two distinct peaks were observed on cytometry histograms and were attributed to gametocyte populations. The activities of established anti-malarial compounds were then measured by flow cytometry and the results were equivalent to those obtained using light microscopy. Primaquine and artemisinin had IC50 of 17.6 μM and 1.0 μM, respectively.Gametocyte sex was apparently distinguishable by flow cytometry as evaluated after induction of exflagellation by xanthurenic acid. These data form the basis of further studies for developing new methods in drug discovery to decrease malaria transmission.

Agelasines J, K, and L from the Solomon Islands Marine Sponge Agelas cf. mauritiana

Three new diterpene alkaloids, agelasine J (3), agelasine K (4), and agelasine L (5), were isolated from the marine sponge Agelas cf. mauritiana collected in the Solomon Islands. The structures of these compounds were elucidated by physical data analyses. They displayed in vitro antimalarial activity against Plasmodium falciparum.

NL MIND-BEST : a web server for ligands and proteins discovery--theoretic-experimental study of proteins of Giardia lamblia and new compounds active against Plasmodium falciparum

There are many protein ligands and/or drugs described with very different affinity to a large number of target proteins or receptors. In this work, we selected Ligands or Drug-target pairs (DTPs/nDTPs) of drugs with high affinity/non-affinity for different targets. Quantitative Structure-Activity Relationships (QSAR) models become a very useful tool in this context to substantially reduce time and resources consuming experiments. Unfortunately most QSAR models predict activity against only one protein target and/or have not been implemented in the form of public web server freely accessible online to the scientific community. To solve this problem, we developed here a multi-target QSAR (mt-QSAR) classifier using the MARCH-INSIDE technique to calculate structural parameters of drug and target plus one Artificial Neuronal Network (ANN) to seek the model. The best ANN model found is a Multi-Layer Perceptron (MLP) with profile MLP 20:20-15-1:1. This MLP classifies correctly 611 out of 678 DTPs (sensitivity=90.12%) and 3083 out of 3408 nDTPs (specificity=90.46%), corresponding to training accuracy=90.41%. The validation of the model was carried out by means of external predicting series. The model classifies correctly 310 out of 338 DTPs (sensitivity=91.72%) and 1527 out of 1674 nDTP (specificity=91.22%) in validation series, corresponding to total accuracy=91.30% for validation series (predictability). This model favorably compares with other ANN models developed in this work and Machine Learning classifiers published before to address the same problem in different aspects. We implemented the present model at web portal Bio-AIMS in the form of an online server called: Non-Linear MARCH-INSIDE Nested Drug-Bank Exploration & Screening Tool (NL MIND-BEST), which is located at URL: http://miaja.tic.udc.es/Bio-AIMS/NL-MIND-BEST.php. This online tool is based on PHP/HTML/Python and MARCH-INSIDE routines. Finally we illustrated two practical uses of this server with two different experiments. In experiment 1, we report by first time Quantum QSAR study, synthesis, characterization, and experimental assay of antiplasmodial and cytotoxic activities of oxoisoaporphine alkaloids derivatives as well as NL MIND-BEST prediction of potential target proteins. In experiment 2, we report sampling, parasite culture, sample preparation, 2-DE, MALDI-TOF, and -TOF/TOF MS, MASCOT search, MM/MD 3D structure modeling, and NL MIND-BEST prediction for different peptides a new protein of the found in the proteome of the human parasite Giardia lamblia, which is promising for anti-parasite drug-targets discovery.

New bioactive halenaquinone derivatives from South Pacific marine sponges of the genus Xestospongia.

Bioassay-directed fractionation of South Pacific marine sponges of the genus Xestospongia has led to the isolation of a number of halenaquinone-type polyketides, including two new derivatives named xestosaprol C methylacetal 7 and orhalquinone 8. Chemical characterization of these two new compounds was achieved by extensive 1D and 2D NMR spectroscopic studies. Evaluation of anti-phospholipase A(2), anti-farnesyltransferase and antiplasmodial activities of this series is presented and structure/activity relationships are discussed. Orhalquinone 8 displayed a significant inhibition of both human and yeast farnesyltransferase enzymes, with IC(50) value of 0.40 microM and was a moderate growth inhibitor of Plasmodium falciparum.

Synthesis and antiplasmodial activity of new indolone N-oxide derivatives.

A series of 66 new indolone-N-oxide derivatives was synthesized with three different methods. Compounds were evaluated for in vitro activity against CQ-sensitive (3D7), CQ-resistant (FcB1), and CQ and pyrimethamine cross-resistant (K1) strains of Plasmodium falciparum (P.f.), as well as for cytotoxic concentration (CC(50)) on MCF7 and KB human tumor cell lines. Compound 26 (5-methoxy-indolone-N-oxide analogue) had the most potent antiplasmodial activity in vitro (<3 nM on FcB1 and = 1.7 nM on 3D7) with a very satisfactory selectivity index (CC(50) MCF7/IC(50) FcB1: 14623; CC(50) KB/IC(50) 3D7: 198823). In in vivo experiments, compound 1 (dioxymethylene derivatives of the indolone-N-oxide) showed the best antiplasmodial activity against Plasmodium berghei, 62% inhibition of the parasitaemia at 30 mg/kg/day.