Transmission of HIV Drug Resistance and the Predicted Effect on Current First-line Regimens in Europe.

BACKGROUND: Numerous studies have shown that baseline drug resistance patterns may influence the outcome of antiretroviral therapy. Therefore, guidelines recommend drug resistance testing to guide the choice of initial regimen. In addition to optimizing individual patient management, these baseline resistance data enable transmitted drug resistance (TDR) to be surveyed for public health purposes. The SPREAD program systematically collects data to gain insight into TDR occurring in Europe since 2001. METHODS: Demographic, clinical, and virological data from 4140 antiretroviral-naive human immunodeficiency virus (HIV)-infected individuals from 26 countries who were newly diagnosed between 2008 and 2010 were analyzed. Evidence of TDR was defined using the WHO list for surveillance of drug resistance mutations. Prevalence of TDR was assessed over time by comparing the results to SPREAD data from 2002 to 2007. Baseline susceptibility to antiretroviral drugs was predicted using the Stanford HIVdb program version 7.0. RESULTS: The overall prevalence of TDR did not change significantly over time and was 8.3% (95% confidence interval, 7.2%-9.5%) in 2008-2010. The most frequent indicators of TDR were nucleoside reverse transcriptase inhibitor (NRTI) mutations (4.5%), followed by nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations (2.9%) and protease inhibitor mutations (2.0%). Baseline mutations were most predictive of reduced susceptibility to initial NNRTI-based regimens: 4.5% and 6.5% of patient isolates were predicted to have resistance to regimens containing efavirenz or rilpivirine, respectively, independent of current NRTI backbones. CONCLUSIONS: Although TDR was highest for NRTIs, the impact of baseline drug resistance patterns on susceptibility was largest for NNRTIs. The prevalence of TDR assessed by epidemiological surveys does not clearly indicate to what degree susceptibility to different drug classes is affected.

Efficacy of tenofovir and efavirenz in combination with lamivudine or emtricitabine in antiretroviral-naive patients in Europe.

BACKGROUND: The combination of tenofovir and efavirenz with either lamivudine or emtricitabine (TELE) has proved to be highly effective in clinical trials for first-line treatment of HIV-1 infection. However, limited data are available on its efficacy in routine clinical practice. METHODS: A multicentre cohort study was performed in therapy-naive patients initiating ART with TELE before July 2009. Efficacy was studied using ITT (missing or switch = failure) and on-treatment (OT) analyses. Genotypic susceptibility scores (GSSs) were determined using the Stanford HIVdb algorithm. RESULTS: Efficacy analysis of 1608 patients showed virological suppression to <50 copies/mL at 48 weeks in 91.5% (OT) and 70.6% (ITT). Almost a quarter of all patients (22.9%) had discontinued TELE at week 48, mainly due to CNS toxicity. Virological failure within 48 weeks was rarely observed (3.3%, n = 53). In multilevel, multivariate analysis, infection with subtype B (P = 0.011), baseline CD4 count <200 cells/mm³ (P < 0.001), GSS <3 (P = 0.002) and use of lamivudine (P < 0.001) were associated with a higher risk of virological failure. After exclusion of patients using co-formulated compounds, virological failure was still more often observed with lamivudine. Following virological failure, three-quarters of patients switched to a PI-based regimen with GSS <3. After 1 year of second-line therapy, viral load was suppressed to <50 copies/mL in 73.5% (OT). CONCLUSIONS: In clinical practice, treatment failure on TELE regimens is relatively frequent due to toxicity. Virological failure is rare and more often observed with lamivudine than with emtricitabine. Following virological failure on TELE, PI-based second-line therapy was often successful despite GSS <3.

Higher proportion of G2P[4] rotaviruses in vaccinated hospitalized cases compared with unvaccinated hospitalized cases, despite high vaccine effectiveness against heterotypic G2P[4] rotaviruses.

The overall vaccine effectiveness of the monovalent rotavirus vaccine in an observational, prospective, multicentre, hospital-based case-control study in Belgium (RotaBel) was 90%. However, rotavirus genotype and co-infecting pathogens are important parameters to take into account when assessing vaccine effectiveness. In this study we specifically investigated the effect of rotavirus genotypes and co-infecting pathogens on vaccine effectiveness of the monovalent vaccine. In addition, we also investigated the effect of co-infecting pathogens on disease severity. From February 2008 to June 2010 stool samples of rotavirus gastroenteritis cases of a random sample of 39 Belgian hospitals were collected and subsequently genotyped. Fisher's exact tests were performed to investigate the relationships between rotavirus genotype, co-infecting pathogens and disease severity. The vaccine effectiveness of a full series of the monovalent rotavirus vaccine against hospitalized rotavirus gastroenteritis caused by G1P[8] rotavirus strains was 95% (95% CI 77.5-98.7). Against G2P[4], the vaccine effectiveness was 85% (95% CI: 63.7-93.8). G4P[8]- and G3P[8]-specific vaccine effectiveness was 90% (95% CI 19.2-98.7) and 87% (95% CI -5.2 to 98.4), respectively. A post-hoc analysis showed that the genotype distribution was significantly related to the vaccination status (p <0.001), whereby G2P[4] strains were proportionally more prevalent in vaccinated cases than in unvaccinated cases. No statistical associations were found between co-infection status and vaccination status, Vesikari severity score or rotavirus genotype. The high vaccine effectiveness against the individual genotypes implies robust protection of the monovalent rotavirus vaccine against hospitalized rotavirus gastroenteritis caused by the major human rotavirus genotypes. The prevalence of G2P[4] requires continued monitoring.

HIV-1 subtype is an independent predictor of reverse transcriptase mutation K65R in HIV-1 patients treated with combination antiretroviral therapy including tenofovir.

Subtype-dependent selection of HIV-1 reverse transcriptase resistance mutation K65R was previously observed in cell culture and small clinical investigations. We compared K65R prevalence across subtypes A, B, C, F, G, and CRF02_AG separately in a cohort of 3,076 patients on combination therapy including tenofovir. K65R selection was significantly higher in HIV-1 subtype C. This could not be explained by clinical and demographic factors in multivariate analysis, suggesting subtype sequence-specific K65R pathways.

Decreasing population selection rates of resistance mutation K65R over time in HIV-1 patients receiving combination therapy including tenofovir.

OBJECTIVES: The use of tenofovir is highly associated with the emergence of mutation K65R, which confers broad resistance to nucleoside/nucleotide analogue reverse transcriptase inhibitors (NRTIs), especially when tenofovir is combined with other NRTIs also selecting for K65R. Although recent HIV-1 treatment guidelines discouraging these combinations resulted in reduced K65R selection with tenofovir, updated information on the impact of currently recommended regimens on the population selection rate of K65R is presently lacking. METHODS: In this study, we evaluated changes over time in the selection rate of resistance mutation K65R in a large population of 2736 HIV-1-infected patients failing combination antiretroviral treatment between 2002 and 2010. RESULTS: The K65R resistance mutation was detected in 144 patients, a prevalence of 5.3%. A large majority of observed K65R cases were explained by the use of tenofovir, reflecting its wide use in clinical practice. However, changing patterns over time in NRTIs accompanying tenofovir resulted in a persistent decreasing probability of K65R selection by tenofovir-based therapy. The currently recommended NRTI combination tenofovir/emtricitabine was associated with a low probability of K65R emergence. For any given dual NRTI combination including tenofovir, higher selection rates of K65R were consistently observed with a non-nucleoside reverse transcriptase inhibitor than with a protease inhibitor as the third agent. DISCUSSION: Our finding of a stable time trend of K65R despite elevated use of tenofovir illustrates increased potency of current HIV-1 therapy including tenofovir.

HIV-1 drug-resistance patterns among patients on failing treatment in a large number of European countries.

BACKGROUND: Information about patterns of HIV-1 drug resistance among treatment-exposed patients is crucial for the development of novel effective drugs. Currently no system exists that monitors patterns of resistance in patients failing therapy. METHODS: The study included 1,988 HIV-1 sequences from patients experiencing therapy failure collected between 2000 and 2004 in 15 European countries. Genotypic resistance was interpreted using the ANRS algorithm. Phenotypic resistance was predicted using the Virco geno- to phenotype system. RESULTS: 80.7% of the sequences included at least one drug-resistance mutation. Mutations were found for NRTIs (73.5%), NNRTIs (48.5%), and protease inhibitors (35.8%). Ninety percent of sequences with genotypic resistance harbored M184V, M41L, K103N, D67N, and/or T215Y. Among NRTIs, resistance was most frequently predicted for lamivudine. About half of all sequences had reduced susceptibility for NNRTIs. Resistance to most boosted protease inhibitors was found in < 25%. No sequence had resistance to all currently available drugs. CONCLUSION: Levels of resistance among patients with therapy failure were high. The patterns of resistance reflect resistance to drugs available for a longer time. Fully suppressive regimens can be designed even for the most mutated HIV because boosted protease inhibitors have remained active against most circulating viruses and new drug classes have become available.

Dietary antioxidants, peroxidation and cardiovascular risks.

Most of the many epidemiological studies in the field strongly suggest that an equilibrated diet such as the so-called "mediterranean diet", is associated with protective effects against major diseases, and particularly, against cardiovascular risks. Since many reports also consider reactive oxygen species or free radical oxidations to be responsible for the accompanying disorders of most pathologies as well as for ageing, it is conceivable that natural plant metabolites such as polyphenols, are likely to play an important role in insuring this protection. Indeed, not only their presence, in particularly high amounts and varieties in foods of such a diet, but also, inter alia, their very potent antioxidant or radical scavenging properties, make polyphenols best accounting for the parodoxical part of the french paradox . Therefore, many efforts have been made to assess the mechanisms for such a cardiovascular disease protection. Whatever convincing were the polyphenols properties demonstrated by many in vitro experiments to support those theories, quite a great number of the results appeared somewhat contradictory when transposed to humans, in the in vivo situation. Some people totally refute this explanation, thinking that health benefits, as far as alcoholic beverages are concerned, originate from ethanol but also, with no doubt, some polyphenols even revealing to be pro-oxidants .

Polyphenols newly extracted in red wine from southwestern France by centrifugal partition chromatography.

Polyphenols from the ethyl acetate extracts of red wine were successfully fractionated using a four-step process (solvent extraction, ion-exchange column chromatography, centrifugal partition chromatography, and semipreparative HPLC), which resulted in the isolation of 22 compounds belonging to different classes of polyphenols (stilbenes, cinnamic acids, flavonoids). Five of them are red wine constituents reported for the first time. The newly isolated compounds include resveratrol dimers, dihydroflavonols, and a cinnamic derivative.

Occurrence of anthocyanin-derived pigments in red wines.

Several anthocyanin-derived pigments that showed UV-visible spectra different from those of the original grape anthocyanins were detected by HPLC-DAD analysis in 1-year-old bottled Port wines from the Douro region. Among these, three malvidin 3-glucoside derived pigments were detected in large amounts, representing approximately 60% of the total anthocyanidin monoglucosides content. These pigments were isolated, purified, and identified by LSI-MS and NMR ((1)H, DQF-COSY, ROESY, HSQC, and HMBC) techniques. The major pigment is malvidin 3-glucoside pyruvic adduct, previously characterized, and the other two corresponded to its respective acetyl and coumaroyl glucoside derivatives. The latter is reported for the first time in red wines.

Stir bar sorptive extraction for the determination of ppq-level traces of organotin compounds in environmental samples with thermal desorption-capillary gas chromatography--ICP mass spectrometry.

The extraction and preconcentration capabilities of a new extraction technique, stir bar sorptive extraction, were combined with the separation power of capillary gas chromatography (CGC) and the low limits of detection (LODs) of inductively coupled plasma mass spectrometry (ICPMS) for the determination of the organotin compounds tributyltin (TBuT) and triphenyltin (TPhT) in aqueous standard solutions, harbor water, and mussels (after digestion with tetramethylammonium hydroxide). Throughout, tripropyltin for TBuT and tricyclohexyltin for TPhT were used as internal standards to correct for variations in the derivatization and extraction efficiency. Calibration was accomplished by means of single standard addition. Derivatization to transform the trisubstituted compounds into sufficiently volatile compounds was carried out with sodium tetraethylborate. The compounds were extracted from their aqueous matrix using a stir bar of 1-cm length, coated with 55 microL of poly(dimethylsiloxane) (PDMS). After 15 min of extraction, the stir bar was desorbed in a thermal desorption unit at 290 degrees C for 15 min, during which the compounds were cold-trapped on a precolumn at -40 degrees C. Flash heating was used to rapidly transfer the compounds to the GC where they were separated on a capillary column with a PDMS coating. After separation, the compounds were transported to the ICP by means of a homemade heated (270 degrees C) transfer line. Monitoring of the 120Sn+ signal by ICPMS during the run of the GC provided extremely low LODs for TPhT in water: 0.1 pg L(-1) (procedure) and 10 fg L(-1) (instrumental) and a repeatability of 12% RSD (n = 10). In harbor water, concentrations of 200 pg L(-1) for TBuT and 22 pg L(-1) for TPhT were found. In fresh mussels, a concentration of 7.2 ng g(-1) (dry weight) TPhT was found. The accuracy of the method was checked by the determination of TPhT in CRM477 (mussel tissue) and comparison of the result to that of an analysis of the same material with a classical liquid/liquid extraction with isooctane.

NMR and simulated annealing investigations of bradykinin in presence of polyphenols.

Epidemiological studies have shown that the incidence of some cardiovascular degenerative diseases appears to be lower in populations with regular but moderate drinking of red wine rich in polyphenols. One of the most important properties of polyphenols is to form complexes with proteins. The linear nonapeptide hormone bradykinin (H-Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9-OH) is involved in a variety of physiological processes such as the cardiovascular processes. Thus, the goal of this work was to study the effects of tannins on the peptide structure by NMR investigations and molecular modeling. The results of these investigations show that in the presence of catechin, the peptide conformation is not affected and is in a random coil structure. On the contrary, the peptide structure is modified by the addition of dimeric proanthocyanidin B3 (catechin 4alpha-->8 catechin). The dimer leads to the formation of a large flexible turn between the 6-9 residues. Thus, the biological activities of bradykinin in the presence of polyphenols could be affected.

DNA triplex stabilization property of natural anthocyanins.

The DNA triplex stabilization property of seven natural anthocyanins (five monoglucosides and two diglucosides) has been measured by the mean of triplex thermal denaturation experiments. We have noticed a difference between the diglucosides that do not modify this melting temperature and the monoglucosides (namely 3-O-beta-D-glucopyranoside of malvidin, peonidin, delphinidin, petunidin and cyanidin) which present a weak but significant stabilizing effect. It appears clearly that the difference between the two series could be due to the supplementary sugar moiety at the 5 position for the diglucosylated compounds, that would make them too crowded to allow interaction with the triplex. Among the monoglucoside series, the most active compounds are the only ones to embody a catechol B-ring in their structure that could be important for such an interaction. The need to have pure and fully characterized compounds to run these measurements, made it possible for us to unambiguously assign the 1H and 13C NMR spectra with the help of 2D NMR experiments. Thus, missing data of compounds not totally described earlier, are provided herein.

Determination of stilbenes (trans-astringin, cis- and trans-piceid, and cis- and trans-resveratrol) in Portuguese wines.

Stilbenes have been shown to have cancer chemopreventive activity and to protect lipoproteins from oxidative damage. A method is described for their direct determination in different types of wine using high-performance liquid chromatography with ultraviolet detection. In a survey of 120 commercial wines from Portugal and France, the highest concentrations of stilbenes were found in red wines. The glucosides of resveratrol were present in higher concentrations than the free isomers. Isolation from wine and characterization of trans-astringin in a large quantity are described for the first time.

Production of 13C-labelled anthocyanins by Vitis vinifera cell suspension cultures.

The use of plant cell cultures for producing isotopically (13C) labelled phenolic substances is reported. Vitis vinifera cells synthesize high levels of anthocyanins when they are cultured in a polyphenol synthesis-inducing medium. Three major anthocyanin monoglucosides found in red wine were identified in grape cells: cyanidin-3-O-beta-glucoside, peonidin-3-O-beta-glucoside, and malvidin-3-O-beta-glucoside. Kinetic study of the intracellular level of phenylalanine and its metabolites showed that it is preferable to add this precursor to grape cell suspensions after the 5th day of culture, i.e. at the beginning of the exponential growth phase. After adding phenylalanine to the culture medium, its uptake was complete and the accumulation of anthocyanins in grape cells was stimulated. Incorporation of [1-13C]-phenylalanine into anthocyanins was measured by means of 13C satellites in the proton NMR spectrum. The maximal rate of 13C enrichment anthocyanins obtained with this technique reached 65%. The production of 13C labelled phenolic compounds was undertaken in order to investigate their absorption and metabolism in humans.

Determination of the uptake of [Pt(NH3)4](NO3)2 by grass cultivated on a sandy loam soil and by cucumber plants, grown hydroponically.

Two cultivation experiments were carried out in order to answer the question to what extent platinum can enter the food chain by accumulation in plants, when the platinum is present in a bio-available form: (i) cucumber plants (Cucumis sativus) were grown hydroponically in nutrient solutions containing [Pt(NH3)4](NO3)2 (from 0.5 to 50 micrograms Pt/l solution); and (ii) a water-soluble platinum compound--[Pt(NH3)4](NO3)2--was added in increasing amounts to a sandy loam soil (from 0.5 to 50 mg Pt/kg soil) and rye grass (Lolium perenne) was grown on it. The roots on the one hand and the green plant fractions in the other hand of the cucumber plants and the rye grass were digested using a high-pressure asher. The platinum concentration was determined by means of a quadrupole-based (VG PQ I) or a double focusing sector field ICP-mass spectrometer (Finnigan MAT, Element), depending on the platinum concentration in the sample solution. The detection limit for platinum obtained with the VG PQ I was observed to be 6 ng/1, while with the 'Element' the detection limit could be improved to 0.5 ng/1 Pt. Accumulation factors were calculated as the ratio of the platinum concentration in the plant to that in the soil or the nutrient solution. The grass grown on spiked soil accumulated platinum only to a slight degree (accumulation factors between 0.008 and 0.032). The hydroponically grown cucumber plants, however, strongly accumulated it (accumulation factors of 11-42 in the shoot and 1700-2100 in the roots). There are three possible causes for the large differences in the accumulation factors: (i) Cucumber plants are dicotyledons; grass, however, is a monocotyledon. Other cultivation experiments already showed that dicotyledons accumulate metals to a higher extent than monocotyledons. (ii) In the grass cultivation experiment, the platinum compound was only added once to the sandy loam soil, namely 2 days before grass was cultivated on it. The nutrient solutions of the cucumber plants were changed twice a week. Consequently, the total amount of platinum that the plants were exposed to during the cultivation of the cucumber plants was higher than during the cultivation of the grass. (iii) Immobilization of the platinum compound in the soil most likely occurred.

Isolation, identification, and antioxidant activity of three stilbene glucosides newly extracted from vitis vinifera cell cultures

Suspension cultures of Vitis vinifera L. (Vitaceae) produce many hydroxylated stilbene glucosides found in red wine. From these cells, we isolated and characterized glycosylated stilbenes, (Z)-piceatannol (3,5,3',4'-tetrahydroxystilbene) -3-O-beta-d-glucopyranoside (6) and (E)- and (Z)-resveratrol (3,5, 4'-trihydroxystilbene)-4'-O-beta-d-glucopyranoside (2 and 7, respectively), which have not previously reported to be constituents of Vitis vinifera or wine. The ability of these compounds to act as radical scavengers was investigated using 1,1 diphenyl-2-picryl-hydrazyl, a stable free radical. Antioxidant activities were assessed by their capacity to prevent Cu2+-induced lipid peroxidation in human low-density lipoprotein.

Quinazoline-2,4(1H,3H)-dione as a substitute for thymine in triple-helix forming oligonucleotides: a reassessment.

A major limitation in triple-helix formation arises from the weak energy of interaction between the third strand and the double-stranded target. We tried to increase the stacking interaction contribution within the third strand by extending the aromatic domain of thymine. We report here the use of 2,4-quinazolinedione as a substitute for thymine in the canonical TA*T triplet. The synthesis and the characterization of the quinazoline beta nucleoside Q and of its phosphoramidite derivative is described. Triple-helix- forming oligonucleotides incorporating Q have been prepared and their ability to form triplexes has been evaluated by UV-monitored thermal denaturation measurements. The introduction of one or multiple Q residues, either contiguous or remote from each other, slightly destabilized triple-stranded structures, whatever the nucleic acid base composition (pyrimidine or GT) of the third strand.

Phenylpropane glycosides from Juniperus phoenicea.

Juniperoside, a new 9-O[beta- D-glucopyranoside]-3,4,5-trimethoxycinnamyl alcohol has been isolated along with the 9- O-[alpha-L-arabinofuranosyl-(1-->6)-beta- D-glucopyranoside]cinnamyl alcohol (rosarin) and coumarin 7- O-beta- D-glucopyranoside (skimmin) from the acetone extract of the aerial parts of Juniperus phoenicea L. The structure elucidation of these natural products was achieved mainly by mass and NMR spectroscopy.

Structural analysis of the mannan region of lipoarabinomannan from Mycobacterium bovis BCG. Heterogeneity in phosphorylation state.

Lipoarabinomannan (LAM) is a major antigen of mycobacterial cell walls, involved in host-Mycobacterium interactions. In a previous work, LAM from the vaccine strain, Mycobacterium bovis BCG, was found to exhibit mannooligosaccharides at its arabinan nonreducing ends (ManLAM). The present report concerns the mannan core structure of this ManLAM. After partial hydrolysis of ManLAM, two populations of mannans (Ma1 and Ma2) were obtained by gel filtration chromatography. Their structural features were defined by means of two-dimensional homo- and heteronuclear (1H-13C) NMR sequences and methylation analysis. They were both found to be composed of an alpha-(1-->6)-linked mannan backbone with alpha-(1-->2)-Manp-linked side chains. They are highly branched, and Ma2 presents a higher frequency of branching than Ma1. Moreover, chemical analysis indicates that only Ma1 is phosphorylated. By a two-dimensional heteronuclear 1H-31P total correlation experiment, the phosphate was found to be involved in a phosphodiester bond between inositol C-1 and glycerol C-3. Then, the molecular mass of mannan was established by mass spectrometry, which revealed a molecular mass of 3517 Da for the major molecular species of Ma1. Likewise, analysis of unfractionated mannans showed the occurrence of other, quantitatively minor molecular species, endowed with two phosphates. This study clearly indicates that the mannan region of M. bovis BCG ManLAM exists as a heterogeneous population of molecules whose structures differ in their degree of glycosylation, level of branching, and phosphorylation state. The hypothesis that the relative abundance of these different molecules modulates the biological functions of LAM is discussed.