A 6-year antifungal stewardship programme in a teaching hospital.

PURPOSE: To describe the antifungal stewardship programme in our hospital and to assess its impact on total antifungal prescriptions and their cost, and on the process of care measures regarding the diagnostic and therapeutic management of invasive aspergillosis and candidaemia. METHODS: We conducted a prospective observational study describing the multifaceted antifungal stewardship programme in place at our French teaching tertiary-care hospital since 2005. Several actions were implemented successively, including the systematic evaluation of all costly antifungal prescriptions (echinocandins, lipid formulations of amphotericin B, posaconazole and voriconazole). RESULTS: A total of 636 antifungal prescriptions were discussed by the antifungal management team from 2005 to 2010 inclusive, mainly from the haematology department (72 %). In 344/636 cases (54 %), a piece of advice was fed back to the physician in charge of the patient, with an 88 % compliance rate. Optimal standard of care was achieved for galactomannan antigen testing, performance of chest computed tomography (CT) scan and voriconazole therapeutic drug monitoring for invasive aspergillosis, with no combination therapies used since 2008. Regarding candidaemia, optimal standard of care was achieved for the timing of antifungal therapy, recommended first-line therapy, duration of therapy and the removal of central venous catheters. Total antifungal prescriptions (in defined daily doses, DDD) and their cost were contained between 2003 and 2010. CONCLUSIONS: The implementation of an antifungal stewardship programme was feasible, sustainable and well accepted. We observed an improved quality of care for some process of care measures, and antifungal use and cost were contained in our hospital.

An antibiotic stewardship program in a French teaching hospital.

OBJECTIVES: An antibiotic stewardship program was implemented in our teaching hospital in 1999, and strengthened in 2005. We report its organization and impact on antibiotic use. METHODS: This observational study was conducted during a 10-year period (2002-2011). RESULTS: Many interventions were implemented: Infectious Diseases Specialists (IDS) led systematic ward rounds in several departments (1999); nominative antibiotic order form (2005); documentation of IDS advice in the patient's electronic medical record (2007); IDS advice triggered by the pharmacist (formulary restriction, 2007) or because of positive blood cultures (2009); automated weekly extraction of advice given into a database (2011). Seven thousand two hundred and five pieces of advice were recorded between 2007 and 2011: 63% following physician request, 26% triggered by the pharmacist and 9% because of positive blood cultures. Advice was provided by IDS in 95% of cases (63% by phone). The number of antibiotic prescriptions remained stable since 2005 at around 400 defined daily doses (DDD)/1000 patient-days. Documenting, sharing, and choice of action were improved due to the database. CONCLUSIONS: Our antibiotic stewardship program is well accepted by physicians and allows controlling antibiotic use in our hospital.

Permanent genetic resources added to Molecular Ecology Resources Database 1 October 2011-30 November 2011.

This article documents the addition of 139 microsatellite marker loci and 90 pairs of single-nucleotide polymorphism sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Aglaoctenus lagotis, Costus pulverulentus, Costus scaber, Culex pipiens, Dascyllus marginatus, Lupinus nanus Benth, Phloeomyzus passerini, Podarcis muralis, Rhododendron rubropilosum Hayata var. taiwanalpinum and Zoarces viviparus. These loci were cross-tested on the following species: Culex quinquefasciatus, Rhododendron pseudochrysanthum Hay. ssp. morii (Hay.) Yamazaki and R. pseudochrysanthum Hayata. This article also documents the addition of 48 sequencing primer pairs and 90 allele-specific primers for Engraulis encrasicolus.

Complex postglacial history of the temperate bark beetle Tomicus piniperda L. (Coleoptera, Scolytinae).

Tomicus piniperda is an economically important pine bark beetle infesting European Pinus spp. stands. We sequenced and analyzed 797 bp of the mitochondrial genome from individuals obtained from 34 populations sampled throughout the European range. We obtained 36 haplotypes, from which a haplotype network was constructed. In the Iberian Peninsula, high-genetic variability was detected with numerous endemic haplotypes. In contrast, the other European populations were less diverse with a single haplotype predominating from the Pyrenees to Scandinavia. Nevertheless, even within Europe, a few populations showed significant amounts of diversity. Four groups were obtained by Spatial Analysis of Molecular Variance, illustrating the regional characteristics of the species. T. piniperda had multiple fragmented refugia in the Iberian Peninsula. These currently isolated populations only partly contributed to postglacial re-colonizations of Northern Europe during interglacials. Nevertheless, few long-range migration events up to Northern Europe were detected, mostly originating from the Pyrenees. In the rest of Europe, the phylogeographical patterns were unclear, because of repeated cycles of contraction and expansion. The genetic analysis showed one glacial refugium in North-Central Europe, whereas other refugia most likely occurred in the Southern Alps, Apennine and the Balkans. The phylogeographical pattern depicted here reflects partly the postglacial history of the beetles' main host tree P. sylvestris.

Antibiotic misuse: a prospective clinical audit in a French university hospital.

The aim of the study presented here was to prospectively audit antibiotic prescriptions given to patients attending L'Archet Hospital in Nice, France, with details of the initial medical examination included in the audit procedure. A total of 122 antibiotic treatments were evaluated, i.e. 31% of all antibiotic therapies initiated in the eight participating departments over the 9-week study period. Forty-two (34%) treatments were found to be unnecessary due to misdiagnosis, and 36 (30%) other treatments were inappropriate. Misdiagnosis, due to the misinterpretation or lack of clinical, microbiological and/or imaging data is thus a major cause of antibiotic misuse. Improvement in the diagnostic process should become part of antibiotic policy.

Comparative population genetic study of two oligophagous insects associated with the same hosts.

A parallel study of the genetic structure of two oligophagous species associated with the same hosts was conducted to determine the main factors shaping the distribution of genetic diversity. The bark beetle Tomicus piniperda and the pine processionary moth (PPM) Thaumetopoea pityocampa are both associated with the genus Pinus and belong to different guilds (xylophagous vs defoliating species). The PPM is an ectophagous species that feeds on the needles of living trees, whereas T. piniperda is endophagous and bores galleries in the inner bark of weakened trees. Both species were sampled in the main regions of France, and their genetic structure was assessed after genotyping with five microsatellite markers. Populations of the PPM were significantly structured. A pattern of isolation by distance was found when distances were calculated as bypassing the Massif Central, whereas no such pattern could be found with raw geographic distances. On the contrary, most populations of T. piniperda were not differentiated. No effect of host species could be detected in either of the two species. We conclude that the two taxa have contrasting effective dispersal rates per generation, and we hypothesize that this reflects the different selection pressures acting on individual fitness via different strategies of host use.

Phylogeographic structure and past history of the circum-Mediterranean species Tomicus destruens Woll. (Coleoptera: Scolytinae).

Phylogeographic studies are often focused on temperate European species with relict footholds in the Mediterranean region. Past climatic oscillations usually induced range contractions and expansions from refugial areas located in southern Europe, and spatial distribution of genetic diversity show that northward expansions were usually pioneer-like. Actually, few studies have focused on circum-Mediterranean species, which probably were not influenced in the same way by climatic oscillations. We present the phylogeography of the bark beetle Tomicus destruens, which is restricted to the whole Mediterranean basin and the Atlantic coasts of North Africa and Portugal. We systematically sequenced 617 bp of the mitochondrial genes COI and COII for 42 populations (N = 219). Analysis revealed 53 haplotypes geographically structured in two clades, namely eastern and western clades, that diverged during the Pleistocene. A contact zone was identified along the Adriatic coast of Italy. Interestingly, we found contrasting levels of genetic structure within each clade. The eastern group was characterized by a significant phylogeographic pattern and low levels of gene flow, whereas the western group barely showed a spatial structure in haplotype distribution. Moreover, the main pine hosts were different between groups, with the Aleppo-brutia complex in the east and the maritime pine in the west. Potential roles of host species, climatic parameters and geographical barriers are discussed and the phylogeographic patterns are compared to classical models of postglacial recolonization in Europe.

Genetic study of the forest pest Tomicus piniperda (Col., Scolytinae) in Yunnan province (China) compared to Europe: new insights for the systematics and evolution of the genus Tomicus.

The pine shoot beetle Tomicus piniperda is present throughout Eurasia. In Europe, it is considered as a secondary pest that rarely causes tree mortality, while heavy damage is observed in Yunnan Province (China) where it exhibits a novel aggregative behaviour during shoot attack. To understand why the ecological characteristics of the European and Chinese populations differ so strongly, we conducted an analysis of population genetic structure on 12 populations in Yunnan and one in JiLin using mitochondrial (COI-COII) and nuclear (ITS2 and 28S rDNA) DNA sequences, and compared the results to those obtained in France. We showed that the Yunnan populations differed markedly from French and JiLin populations. For all three markers, the genetic distances measured between the Tomicus from Yunnan and those from France were similar to distances previously observed between species. Similar distances were found between Yunnan and JiLin populations. Conversely, the distances between French and JiLin individuals were substantially lower, falling in the intraspecific range. We concluded that the individuals sampled in Yunnan belong to a new, undescribed species (Tomicus sp. nov.). We also showed that some individuals belong to the species T. brevipilosus that had never been recorded from this region before. Evolution of the genus Tomicus is discussed in the light of these new results.

Effects of drought stress and high density stem inoculations with Leptographium wingfieldii on hydraulic properties of young Scots pine trees.

We examined drought-induced changes in susceptibility of potted Scots pine (Pinus sylvestris L.) trees to a bark-beetle associated fungus (Leptographium wingfieldii Morelet, from the bark beetle Tomicus piniperda L.). Five-year-old field-grown trees were transplanted to 50-l pots and grown for 1 year before the treatments were applied. Trees in the drought treatment were subjected to several successive, 3-week-long drought cycles, with predawn water potential dropping below -2 MPa at peak drought intensity. The experimental drought cycles were more severe than the natural drought episodes usually recorded in Scots pine stands. Trees were then mass-inoculated with L. wingfieldii at a density close to the critical threshold density of inoculations (400 m(-2)) above which tree resistance is overcome. Inoculation of well-watered trees resulted in induced reaction zones around the inoculation points and very limited damage (resinosis) in the sapwood. Drought alone had no long-lasting consequences on tree water relations, except for a decrease in hydraulic conductance in the youngest segments of the main stem. However, the combination of mass-inoculation and drought stress after inoculation resulted in a dramatic loss of stem hydraulic conductivity that was paralleled by conspicuous damage to the sapwood (resinosis, drying and blue staining). There was a close correlation between amount of visible sapwood damage and loss of hydraulic conductivity. The intensity of induced reactions in the phloem was unaffected by drought stress. We conclude that tree defence against L. wingfieldii is decreased during severe drought stress, resulting in changes in the spread and action of the fungus in the sapwood but not in the phloem.

Gene induction of stilbene biosynthesis in Scots pine in response to ozone treatment, wounding, and fungal infection.

The S-adenosyl-L-methionine:pinosylvin-O-methyltransferase (PMT) gene was sequenced from Scots pine (Pinus sylvestris). The open reading frame is arranged in two exons spaced by one 102-bp intron. Promoter regulatory elements such as two "CAAT" boxes and one "TATA" box were identified. Several cis-regulatory elements were recognized: stress-responsive elements (Myb-responsive elements) as well as G, H, and GC boxes. Moreover, elicitor-responsive elements (W boxes) and a sequence resembling the simian virus 40 enhancer core were found. In phloem and needles of control trees, the transcripts of stilbene synthase (STS) and PMT were hardly detectable. Increased ozone fumigation up to 0.3 microL L(-1) enhanced the transcript level of STS and PMT in needles but not in healthy phloem. Wounding, e.g. mock inoculation, of stem-phloem was characterized by a transient increase in STS and PMT transcripts, which was more pronounced in the case of fungal inoculation. Combination of fungal-challenge or mock treatment with ozone resulted in a positive interaction at 0.3 microL L(-1). Scots pine stilbene formation appeared to be induced via STS and PMT gene expression upon ozone and fungal stress as well as wounding. The broad stress-responsiveness is in agreement with the range of various cis-acting elements detected in the STS and PMT promoters.

Mechanical injury and fungal infection induce acquired resistance in Norway spruce.

Norway spruce trees (Picea abies (L.) Karst.) pretreated by wounding and fungal infection showed highly enhanced resistance to a subsequent challenge inoculation with the pathogenic bluestain fungus Ceratocystis polonica (Siem.) C. Moreau. This is the first time the effectiveness of the constitutive and inducible defenses has been shown to depend on prior wounding and infection in conifers, although such acquired resistance has previously been found in several angiosperms. Trees that were pretreated with a combination of 12 bark wounds (1.6 x 10 cm), four fungal inoculations and four sterile inoculations 1-15 days before mass inoculation with C. polonica at 400 inoculations per square meter over a 0.8 m stem section had significantly shorter necroses in the phloem, less bluestained sapwood, and less dead cambium than untreated control trees. Pretreatment with four fungal or sterile inoculations alone did not lead to enhanced resistance. Pretreatment by bark wounding alone seemed to provide an intermediate degree of resistance compared to bark wounding, fungal inoculations and sterile inoculations combined. All trees had a marked increase in the number of resin ducts in the year of inoculation compared with previous years, suggesting that formation of traumatic resin ducts play an important role in the development and maintainance of enhanced resistance.

Induced Responses in Phenolic Metabolism in Two Norway Spruce Clones after Wounding and Inoculations with Ophiostoma polonicum, a Bark Beetle-Associated Fungus.

Two Norway spruce (Picea abies Karst.) clones, one resistant and the other susceptible to mass inoculation with Ophiostoma polonicum Siem., were compared with regard to their phenolic compositions and chalcone synthase (CHS) and stilbene synthase activities of their phloem before and at 6 and 12 d after artificial inoculation with sterile malt agar or O. polonicum. In unwounded phloem, the resistant clone differed from the susceptible clone by the presence of taxifolin glycoside, lower concentrations of stilbene glycosides, and higher CHS activity. After inoculation, (+)-catechin concentration and CHS activity dramatically increased around the wound, particularly in the resistant clone. Stilbene synthase activity also increased, but more slowly and to a lower level, whereas the concentrations of stilbenes remained stable. Tanning ability decreased in the susceptible clone, whereas it remained stable in the resistant one. It is proposed that the induced phenolic response of Norway spruce phloem consists of an activation of the phenolic pathway, finally leading to tannins and insoluble polymers. It is suggested that resistance to O. polonicum depends on the ability of the tree to easily activate the flavonoid pathway.

[Parasitism of Ips sexdentatus (Insecta: Scolytidae) by Parasitorhabditis ipsophila (Nematoda: Rhabditidae)]. / Observations sur le parasitisme d'Ips sexdentatus (Insecta: Scolytidae) par Parasitorhabditis ipsophila (Nematoda: Rhabditidae).

The study of parasitism percentages and contaminations intensity in Ips sexdentatus parasitized by P. ipsophila as well as the examination of the wormholes in the galleries of the bark beetle gave better insight into certain features of nematode biology. Larvae of I. sexdentatus could be infected, whereas pupae could not. Adults were contaminated from initial stages of maturation and throughout preswarming maturation. Following swarming and installation on a new tree, insects were rapidly decontaminated, but recontamination could occur by the end of oviposition. P. ipsophila larvae were found in the mesenteron before they penetrated into the hind gut. Seemingly, the parasite underwent no evolution within its host. All developmental stages of the nematode could be observed in the galleries of the bark beetle as long as the latter was present. No apparent relation exists between parasitism of the digestive tract by P. ipsophila and parasitism of the body or fat body by Parasitaphelenchus or Contortylenchus diplogaster. P. ipsophila exerts very limited effects on I. sexdentatus populations. A slight delay in swarming and initiation of oviposition, and a very low decrease in density of notches of oviposition and of eggs was observed, but the features of the gallery of oviposition (total length, length before the first notch) showed no alteration. No mortality was detected.

[Disturbances in the digestive tract of Ips sexdentatus (Insecta: Scolitidae) induced by Parasitorhabditis ipsophila (Nematoda: Rhabditidae)]. / Les perturbations induites par Parasitorhabditis Ipsophila (Nematoda: Rhabditidae) dans le tube digestif d'Ips sexdentatus (Insecta: Scolytidae).

The histological study of the digestive tract in Ips sexdentatus parasitized by P. ipsophila showed major destruction of the microvillosities as well as vacuolization of the epithelial cells in the terminal part of the mesenteron. No damage could be observed in the hind gut nor during low contamination. Concurrently, the biochemical study indicated a lower amount of proteins in the alimentary bolus of parasitized insects along with a higher amount of proteins in the intestinal wall. Damage seemed chiefly mechanical and accounted for the disorders previously observed in populations. The apparent contradiction between the magnitude of damage to the intestinal epithelium and the mild effects of parasitism on host populations might be explained by the preferential location of nematodes in the terminal part of the mesenteron and the proximal part of the hind gut.

[Histological observations of the capsules induced by Contortylenchus diplogaster v. Lins (Nematoda: Allantonematidae) in the adipose tissue of Ips sexdentatus Boern. (Coleoptera: Scolytidae)]. / Observations histologiques sur les capsules induites par Contortylenchus diplogaster v. Lins. (Nematoda: Allantonematidae) dans le tissu adipeux d'Ips sexdentatus Boern. (Coleoptera: Scolytidae).

The evolution of the capsules induced by C. diplogaster in the fat body of I. sexdentatus is followed by histological methods from the pupal stage to the oviposition of the host. Early in the pupal stage of the insect, the parasitic female of C. diplogaster has not oviposited yet and the nuclei of the capsule that surrounds it are gathered in tetrads; some of these nuclei are in a well forwarded degenerescence. These observations suggest that the syncytial capsule should result both from an amitotic division of the nuclei followed by a degenerescence of some of them and from a fusion of cells. Late in the pupal stage, the nuclei are set apart from each other and distributed at the periphery of the capsule. The oviposition of the nematode takes place when the host is an adult. After the development of the larvae has been made into the capsule, this one ends in breaking, generally when the bark beetle has attacked a new host-tree. Then, the larvae of the nematode leave the insect and invade the galleries. The parasitic development of C. diplogaster always takes place quite in the fat body of the beetle.

[Weight variations of adipose tissue and ovaries and variations in the length of ovocytes in Ips sexdentatus Boern (Coleoptera: Scolytidae); relation to parasitism by nematodes]. / Les variations pondérales du tissue adipeux et des ovaires, et les variations de longueur des ovocytes, chez Ips sexdentatus Boern (Coleoptera: Scolytidae); relations avec le parasitisme par les nématodes.

The variations of the weight of the fat body and the ovaries, as well as the length of the maturest oocyte, have been studied in Ips sexdentatus, from the end of the maturation of pre-swarming to the 24th hour after the copulation. In the healthy Insects, the weight of the ovaries and the size of the terminal oocyte grow till the swarming, but decline slightly at the 3rd hour after the copulation; the growth recovers afterwards, at first slow, then very steep after the 12th hour. The fat body declines at the swarming, but grows distinctly at the 6th hour; its weight decreases strongly at the 12th and 18th hour, then grows again considerably at the 24th hour. The variations of the weight of the ovaries (and of the size of the terminal oocyte) may be compared with that of the fat body, if we take into consideration the being of a delay in the "response" of the ovaries. This combined variations might be agreed with important periods of the biology of the bark-beetle: swarming, slow then steep maturation of the ovaries, oviposition. In the Insects parasitized by the Nematodes Parasitaphelenchus sp., P. sexdentati (Fuchs 1937) Rühm 1956, Contortylenchus diplogaster (V. Lins 1890) Rühm 1956, the fat body and the ovaries are always slighter, and the terminal oocyte less developed than in the healthy Insects. Moreover, the fat body of the infected Insects cannot accumulate the indispensable stocks for the maturation of the oocytes, just as these one seem to want that. Therefore the maturation of the oocytes of this Insects is delayed. This delay, by accumulating itself for the successive oocytes, might explain the important reduction of the oviposition in the parasitized Insects. The effect of the Nematodes on the ovaries of Ips sexdentatus might be indirect, the direct effect being related to the fat body or to the blood. On the otherside, the abdominal fat body seems to be more affected by the parasitism than the prothoracic one.

[Cellular encapsulation of Contortylenchus diplogaster v. Linser. (Nematoda: Allantonematidae) in the fat body of Ips sexdentatus Boern. (Coleoptera: Scolytidae) (author's transl)]. / Encapsulement cellulaire de Contortylenchus diplogaster v. Linser. (Nematoda: Allantonematidae) dans le tissu apideux d'Ips sexdentatus Boern. (Coleoptera: Scolytidae).

The parasitic females of C. diplogaster may induce the formation of cellular capsules in I. sexdentatus. This capsules may be free in the body cavity or attached to the fat body of the Insect. Each of them contains one parasitic female accompanied by its eggs and its larvae. They correspond to hypertrophied fat body cells, which become a syncytium. The larvae of Nematode which are inside the capsule, feed on this, and the older capsules are only bags filled with larvae, which will end by breaking themselves and by setting free their content in the body cavity of the host. The phenomenon, here observed in a Tylenchoïdea of which the Insect is the sole host, seems widely distributed in parasitic Nematodes of Insects, for it is already known in the Spiruroïdea and Filarioïdea. Otherwise, while the parasitic adults of all other species of Allantonematidae are considered to be free in the body cavity of their host, C. diplogaster may have a part of its life cycle inside the fat body of the host.