Implementation of a simple innovative system for postprescription antibiotic review based on computerized tools with shared access.

BACKGROUND: Controlling antibiotic use in healthcare establishments limits their consumption and the emergence of bacterial resistance. AIM: To evaluate the efficiency of an innovative antibiotic stewardship strategy implemented over three years in a university hospital. METHODS: An antimicrobial multi-disciplinary team (AMT) [pharmacist, microbiologist and infectious disease specialist (IDS)] conducted a postprescription review. Specific coding of targeted antibiotics (including broad-spectrum ß-lactams, glycopeptides, lipopeptides, fluoroquinolones and carbapenems) in the computerized physician order entry allowed recording of all new prescriptions. The data [patient, antibiotic(s), prescription start date, etc.] were registered on an AMT spreadsheet with shared access, where the microbiologist's opinion on the drug choice, based on available microbiology results, was entered. When the microbiologist and pharmacist did not approve the antibiotic prescribed, a same-day alert was generated and sent to the IDS. That alert led the IDS to re-evaluate the treatment. FINDINGS: From 2012 to 2014, 2106 targeted antibiotic prescriptions were reviewed. Among them, 389 (18.5%) generated an alert and 293 (13.9%) were re-evaluated by the IDS. Recommendations (mostly de-escalation or discontinuation) were necessary for 136 (46.4%) and the prescribers' acceptance rate was 97%. The estimated intervention time was <30 min/day for each AMT member. This system allowed correct use of targeted antibiotics for 91.8% of prescriptions, but had no significant impact on targeted antibiotic consumption. CONCLUSION: This computerized, shared access, antibiotic stewardship strategy seems to be time saving, and effectively limited misuse of broad-spectrum antibiotics.

Predicting economic losses from the continued spread of the Mexican rice borer (Lepidoptera: Crambidae).

The Mexican rice borer, Eoreuma loftini (Dyar) (Lepidoptera: Crambidae), is an invasive species that originated from Mexico, and it is threatening to cause major economic losses to sugarcane, Saccharum spp., and rice, Oryza sativa L., industries in Louisiana. The insect is expected to reach sugarcane and rice production areas in Louisiana by 2008, and infest all of Louisiana sugarcane and rice industries by 2035. When all sugarcane in Louisiana becomes infested, annual yield losses of $220 million would be expected for a cultivar of comparable susceptibility to LCP 85-384 (assuming this cultivar is planted on 100% of the production area). This also assumes the use of the current practice of rainfed production and one application of insecticide, which is presently used by farmers in Louisiana. Irrigation with 30 cm of water is predicted to reduce estimated losses by 29%, whereas four applications of a biorational insecticide such as tebufenozide are expected to reduce the loss in revenue by 53%. The use of the resistant 'HoCP 85-845' would reduce the projected loss in revenue by 24%. Combining all three management tactics on sugarcane, anticipated net loss in revenue would decrease by 66%. The rice industry in Louisiana is projected to suffer from a loss in revenue of $45 million when the entire state is infested. A 77% reduction in loss in revenue is expected with one application of lambda-cyhalothrin. A quarantine on east Texas sugarcane is estimated to save the Louisiana industry between $1.1 billion and $3.2 billion (depending on management) during the time needed for the insect to fully invade the state's sugarcane and rice producing area by natural migration rather than by accidental introduction. The rapid deployment of appropriate management tactics will have a key role in reducing the anticipated economic impact of E. loftini once it becomes a pest in Louisiana sugarcane and rice.

Resistance to the Mexican rice borer (Lepidoptera: Crambidae) among Louisiana and Texas sugarcane cultivars.

A 2-yr study to evaluate Louisiana and Texas sugarcane, Saccharum spp., cultivars for resistance to the Mexican rice borer, Eoreuma loftini (Dyar) was conducted in two locations in Texas, chosen for having different infestation levels. Criteria for assessment of resistance included percentage of bored internodes and adult emergence holes, the latter used to determine the relative impact of each cultivar on the potential areawide buildup or reduction of adult E. loftini populations. A recently released cultivar, HoCP 85-845, seemed to lose a portion of its resistance under heavy E. loftini infestation pressure, suggesting its value only in moderate-to-low infestation conditions. Cultivar CP 70-321 was the most resistant. Results indicated that cultivar LCP 85-384 was significantly (P < 0.05) more susceptible than NCo 310, traditionally the most susceptible cultivar commercially produced in Texas. In 2001, LCP 85-384, which now represents 85% of the production area in Louisiana, had the greatest moth production per hectare (17,052 +/- 3,956) under the lower infestation pressure, significantly (P < 0.05) higher than HoCP 85-845 (3,038 +/- 2,353). In a portion of the test at the high-infestation location, high levels of sodium and magnesium salt stress (15-30-cm soil depth) were associated with higher E. loftini damage in all cultivars except HoCP 91-555 and CP 70-321.

Time-resolved fluorescence imaging of slab gels for lifetime base-calling in DNA sequencing applications.

A compact time-resolved near-IR fluorescence imager was constructed to obtain lifetime and intensity images of DNA sequencing slab gels. The scanner consisted of a microscope body with f/1.2 relay optics onto which was mounted a pulsed diode laser (repetition rate 80 MHz, lasing wavelength 680 nm, average power 5 mW), filtering optics, and a large photoactive area (diameter 500 microns) single-photon avalanche diode that was actively quenched to provide a large dynamic operating range. The time-resolved data were processed using electronics configured in a conventional time-correlated single-photon-counting format with all of the counting hardware situated on a PC card resident on the computer bus. The microscope head produced a timing response of 450 ps (fwhm) in a scanning mode, allowing the measurement of subnano-second lifetimes. The time-resolved microscope head was placed in an automated DNA sequencer and translated across a 21-cm-wide gel plate in approximately 6 s (scan rate 3.5 cm/s) with an accumulation time per pixel of 10 ms. The sampling frequency was 0.17 Hz (duty cycle 0.0017), sufficient to prevent signal aliasing during the electrophoresis separation. Software (written in Visual Basic) allowed acquisition of both the intensity image and lifetime analysis of DNA bands migrating through the gel in real time. Using a dual-labeling (IRD700 and Cy5.5 labeling dyes)/two-lane sequencing strategy, we successfully read 670 bases of a control M13mp18 ssDNA template using lifetime identification. Comparison of the reconstructed sequence with the known sequence of the phage indicated the number of miscalls was only 2, producing an error rate of approximately 0.3% (identification accuracy 99.7%). The lifetimes were calculated using maximum likelihood estimators and allowed on-line determinations with high precision, even when short integration times were used to construct the decay profiles. Comparison of the lifetime base calling to a single-dye/four-lane sequencing strategy indicated similar results in terms of miscalls, but reduced insertion and deletion errors using lifetime identification methods, improving the overall read accuracy.

An assessment of the phylogenetic relationship among sugarcane and related taxa based on the nucleotide sequence of 5S rRNA intergenic spacers.

5S rRNA intergenic spacers were amplified from two elite sugarcane (Saccharum hybrids) cultivars and their related taxa by polymerase chain reaction (PCR) with 5S rDNA consensus primers. Resulting PCR products were uniform in length from each accession but exhibited some degree of length variation among the sugarcane accessions and related taxa. These PCR products did not always cross hybridize in Southern blot hybridization experiments. These PCR products were cloned into a commercial plasmid vector PCR 2.1 and sequenced. Direct sequencing of cloned PCR products revealed spacer length of 231-237 bp for S. officinarum, 233-237 for sugarcane cultivars, 228-238 bp for S. spontaneum, 239-252 bp for S. giganteum, 385-410 bp for Erianthus spp., 226-230 bp for Miscanthus sinensis Zebra, 206-207 bp for M. sinensis IMP 3057, 207-209 bp for Sorghum bicolor, and 247-249 bp for Zea mays. Nucleotide sequence polymorphism were found at both the segment and single nucleotide level. A consensus sequence for each taxon was obtained by Align X. Multiple sequences were aligned and phylogenetic trees constructed using Align X. CLUSTAL and DNAMAN programs. In general, accessions of the following taxa tended to group together to form distinct clusters: S. giganteum, Erianthus spp., M. sinensis, S. bicolor, and Z. mays. However, the two S. officinarum clones and two sugarcane cultivars did not form distinct clusters but interrelated within the S. spontaneum cluster. The disclosure of these 5S rRNA intergenic spacer sequences will facilitate marker-assisted breeding in sugarcane.

Development of Polymerase Chain Reaction Primers Highly Specific for Xanthomonas albilineans, the Causal Bacterium of Sugarcane Leaf Scald Disease.

New primers were developed that greatly improved the specificity of the polymerase chain reaction (PCR) protocol for Xanthomonas albilineans, the causal agent of sugarcane leaf scald disease. Length-polymorphic PCR products, amplified under the current PCR protocol from the 16S-23S ribosomal DNA intergenic transcribed spacers (ITS) of X. albilineans and three unidentified sugarcane saprophytic bacterial species, were cloned and sequenced. Fourteen other nonredundant ITS sequences retrieved from the database were highly homologous to the sequence of X. albilineans. Two X. albilineans-specific PCR primers, namely, PGBL1 (5' CTT TGG GTC TGT AGC TCA GG) and PGBL2 (5' GCC TCA AGG TCA TAT TCA GC), were designed based on a multiple sequence alignment among these 18 sequences. These two primers permitted specific PCR amplification of a 288-bp DNA product from all 71 diverse X. albilineans strains tested. No amplification product was observed from any other bacterial species tested, including the three unidentified sugarcane saprophytes. The new PCR protocol has been routinely used to detect the leaf scald pathogen from infected sugarcane tissues.

Ultrasensitive near-infrared laser-induced fluorescence detection in capillary electrophoresis using a diode laser and avalanche photodiode.

A sensitive fluorescence detector for capillary electrophoresis consisting of a semiconductor near-infrared diode laser and a single photon avalanche diode (SPAD) is described. The sensitivity of this system was demonstrated by the separation and analysis of four tricarbocyanine dyes using capillary electrophoresis and a running buffer consisting of 98% methanol and 2% water with 40 mM borate (pH 9.4). The LOD for the dye, IR-132, was found to be 4.41 zmol with the dynamic range found to be approximately four orders of magnitude in concentration. Based on the sampling volume of the system, the number of molecules actually detected at this LOD was approximately 27. To further demonstrate the utility of this diode-based detector, various amino acids were derivatized with a highly anionic near-IR labelling dye. The conjugates were separated in a running buffer comprised of predominately methanol and a cationic surfactant added to reverse the electroosmotic flow. The LOD values for various amino acids were found to be in the low zmol range.

On-line fluorescence lifetime determinations in capillary electrophoresis.

A near-infrared time-correlated single-photon counting instrument was developed for on-line fluorescence lifetime determinations of components separated by capillary electrophoresis (CE). The lifetimes of the migrating components were determined using maximum likelihood estimators, which are computationally easy to perform and yield values with high precision and favorable accuracies in the limit of low photocounts within the decay profile. The laser source used in the present system was a passively mode-locked Ti-sapphire laser with a single-photon avalanche diode serving as the fast detector. The instrument response function of this system was determined to be 165 ps (fwhm). Electrophorectic separation of two near-IR dyes, DTTCI (cationic) and IR-125 (anionic), in a 95:5 methanol/water running buffer (pH = 9.5) produced electrophoretic peak widths at the base of approximately 1 -9 s, which set the integration time for collection of the decay profiles. At a loading level of 1.42 zmol for IR-125 and 49 zmol for DTTCI, lifetime values were determined to be 482 +/- 14 ps for IR-125 and 943 +/- 23 ps for DTTCI, which agreed favorably with the lifetimes determined for these dyes using static measurements at high concentrations. To minimize background resulting from scattered photons in ultradilute conditions, which introduces bias into the lifetime determination, the calculation was initiated at a fixed time delay with respect to the excitation pulse. To demonstrate the feasibility of making lifetime determinations in capillary gel electrophoresis, where the gel can produce high scattering backgrounds, the lifetimes of C-terminated fragments produced from the M13mp 18 template and labeled at the 5' end of a universal M13 sequencing primer with a near-IR fluorescent tag were determined. The collection of lifetimes for 30 different peaks in the electropherogram yielded a mean value of 581 ps and a standard deviation of +/- 9 ps.

Human serum stimulates the production of G-CSF, IL-1, IL-6 and IL-8 by human peripheral blood leucocytes.

Human serum induces human peripheral blood leucocytes (PBL) to release an activity stimulating neutrophil colony formation (G-CSA) from human bone marrow cells. By titrating individual growth factors and using specific neutralizing antibodies we showed that: human serum contains very low levels of G-CSF which are by themselves insufficient to stimulate myeloid colony formation in primary human bone marrow cultures and cannot account for the serum releaser activity; that although no detectable levels of IL-1, IL-2, IL-3, IL-4, IL-6 or IL-8 are found in the serum, anti IL-1 antibodies partially block the release of G-CSA when added early during PBL incubation; that PBL incubated in the absence of serum for 2 d produce small amounts of IL-1, IL-6, IL-8 and G-CSF and this is increased 6-16 fold in the presence of human serum; and that the neutrophil colony-stimulating activity released by PBL incubated with human serum is G-CSF.

Induction of swelling, synovial hyperplasia and cartilage proteoglycan loss upon intra-articular injection of transforming growth factor beta-2 in the rabbit.

Transforming growth factor beta (TGF-beta) is a multifunctional homodimeric polypeptide with potent actions upon many target cells, including those of mesenchymal and haemopoietic lineage. The recent reports of high levels of the cytokine in rheumatoid synovium and synovial fluid, prompted this study into the effect of intra-articular injection of TGF beta-2 into rabbit knee-joints. Four daily injections of 1 microgram caused swelling, probably as a consequence of prostaglandin E2 production, synovial fibroblastic hyperplasia and a striking loss of femoral condyle proteoglycan. Using the polymerase chain reaction, no evidence could be obtained for the induction of interleukin-1 alpha gene expression in either synovial tissue or synovial fluid cells. These findings suggest that the TGF-beta present in the rheumatoid joint may contribute directly to the pathogenesis of rheumatoid arthritis.

Polymorphism of spironolactone.

Single crystals of two polymorphic and four solvated crystalline forms of spironolactone (C24H32O4S) were obtained using different solvents. The morphology, symmetry, and crystallographic parameters were determined for all crystal forms except for the one obtained from methanol. The stability and transformation of each type of crystal were studied by DSC, TGA, and X-ray diffraction analysis. The structural data of three forms allowed the observation of the change of conformation of the molecules of spironolactone in the three different lattices.

Calcitonin-like immunoreactivity and calcitonin gene expression in the placenta and in the mammary gland of the rat.

Recently, the presence of monomeric CT in plasma and milk was reported by others in a lactating woman surgically thyroidectomized. Similarly, the placenta was thought to be a possible source of CT. Since such findings were based exclusively on immunological arguments, we have investigated the CT gene expression in these rat tissues. CT mRNAs were detected by dot-blot hybridization of total RNAs extracted from rat tissues with a 32P-labelled human CT cDNA probe. Subcellular fractions of each tissue were screened for CT-like immunoreactivity using two different antibodies. With one antibody, extracts of the mammary gland and placenta both produced full displacement of labelled human CT from the antiserum and serial dilutions of the extracts gave displacement curves parallel to that of synthetic human CT, which suggests immunological similarity. However, dilution curves were not parallel for the second antibody, and for both antisera, CT-like immunoreactivity was found in all subsellular fractions from nuclei to cytosols. Immunoprecipitation of translation products from poly (A)+RNAs of placenta showed two major bands around 30 kD. Under stringent conditions, the weak hybridization of placental RNAs seen by dot-blot under less stringent conditions disappeared. Northern analyses of total RNAs from the placenta failed to detect mRNA of 1 k base size like in thyroid glands, but hybridization under weak stringent conditions occurred with larger mRNAs (around 4.4 and 2.4 k bases). Immunoprecipitation of translation products from mRNAs of rat mammary glands showed three major bands around 46, 30 and 20 kD. Dot-blot hybridization of total RNAs extracted from mammary glands was also negative.(ABSTRACT TRUNCATED AT 250 WORDS)

1,25-Dihydroxycholecalciferol effects on plasma calcitonin levels and calcitonin mRNA in normal or partially vitamin D-depleted rats.

The physiological effect of 1,25-(OH)2D3 on the regulation of calcitonin (CT) secretion was studied by measuring plasma CT levels and CT mRNAs extracted from thyroid glands of normal (D+) or partially vitamin D-depleted rats (D-). In both groups, acute 1,25-(OH)2D3 administration of 0.1 microgram/kg b.w. yielded an early drop in plasma calcium concentrations (around 0.6-1 mg/dl) with a maximum decrease 15 min after treatment. In spite of this hypocalcemia, a significant rise in plasma CT levels was observed within 5 min in D+ animals and within 30 min in D- animals after injection of the vitamin D metabolite. Nevertheless, the increased CT secretion was not associated with a marked and sustained rise in CT mRNA levels measured by dot-blot hybridization or CT mRNA activity evaluated by translation assay. By contrast to the observations made previously using supra-physiological doses of the vitamin D metabolites, no clear-cut effect on CT mRNA levels was found with lower doses. If we hypothesized that 1,25-(OH)2D3 plays a physiological role in CT secretion, our results suggest that this rapid control could be exerted at a post-translational level may be via an increase in the cytoplasmic ionized calcium concentration of C-cells.

Calcitonin mRNA activity in genetically obese rats.

The etiology of the alterations in calcitonin (CT) secretion and plasma calcium of genetically obese (fa/fa) rats is unknown. In this study, we tested the postulate that there is an early occurrence of abnormalities in CT biosynthesis by thyroid glands of these rodents. Male genetically obese Zucker (fa/fa) rats and their lean littermates were therefore studied from 30 days to 10 months of age. Obese animals were characterized by hypercalcemia (delta approximately equal to 1 mg/dl), already present at 30 days of age. Increased thyroidal CT stores began at 6 weeks of age in fatty rats. Plasma CT levels were decreased in obese animals from 30 days to 10 weeks of age and were not different in leans and fatties 2 weeks later, but were higher at 10 months in fatty rats. Poly A + RNA were extracted from thyroid glands and subjected to translation assays. After SDS-PAGE, specific immunoprecipitates were autoradiographed and quantified by integration. A similar translation product with an apparent mol wt of 15,000 was specifically immunoprecipitated with CT antisera in obese (fa/fa) and lean Zucker rats at different ages. In 30-day-old fatty rats, a 50% decrease in translatable CT mRNA was observed in association with decreased plasma CT levels. In 12-week-old fatty rats, the translatable CT mRNA activity was unchanged or higher when compared to lean littermates, and clearly higher in 10-month-old fatty rats. The CT mRNA levels measured by dot-blot or northern blot hybridization paralleled at each stage studied the CT mRNA activity, determined by translation. It was concluded that in basal conditions, plasma CT level variations during development reflect the biosynthetic activity of C cells in genetically obese rats. The data presented in this study strengthen the point of an early occurrence of abnormalities in CT mRNA activity and in plasma calcium of fa/fa rats.

Increased level of calcitonin mRNA after 1,25-dihydroxyvitamin D3 injection in the rat.

Vitamin D metabolites are able to change plasma calcitonin (CT) levels, but nothing is known about a possible effect at the CT gene level. Here we have investigated the acute effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on the CT biosynthetic activity of thyroid glands from adult rats. Plasma CT levels were significantly increased (X2) 1 and 2 h after 1,25-(OH)2D3 injection in the face of unchanged plasma calcium values. The thyroidal CT content also was unchanged. A 2-fold increase in CT mRNA level measured by dot-blot hybridization occurred 1 and 2 h after 1,25-(OH)2D3 administration. Expression of CT gene products was examined in the rabbit reticulocyte lysate cell-free translation assay. After polyacrylamide gel electrophoresis, specific immunoprecipitates were autoradiographed and quantified by integration. A single precursor of Mr approximately equal to 15 000 could be specifically immunoprecipitated with CT antisera. A 3-4-fold rise in translatable CT mRNA activity was observed 1 and 2 h after 1,25-(OH)2D3 injection. Thus, parallel changes in CT mRNA level, CT mRNA activity and plasma CT levels were observed in adult female rats after administration of 1,25-(OH)2D3. These findings demonstrate for the first time that 1,25-(OH)2D3 enhanced CT gene expression in the face of unchanged plasma calcium levels.