S-metolachlor (S-MET) are two herbicides widely used, often as mixtures. The present work examined whether the presence of S-MET affects the ATZ-biodegradation activity of the bioaugmentation bacterium Pseudomonas sp. strain ADP in a crop soil. S-MET concentrations were selected for their relevance in worst-case scenarios of soil contamination by a commercial formulation containing both herbicides. At concentrations representative of application of high doses of the formulation ), the presence of pure S-MET significantly affected neither bacteria survival  nor its ATZ-mineralization activity. Consistently, biodegradation experiments, in larger soil microcosms spiked with 20× or 50×RD of the double formulation and inoculated with the bacterium, revealed ATZ to be rapidly (in up to 5 days) and extensively (>96%) removed from the soil. During the 5 days, concentration of S-MET decreased moderately to about 60% of the initial, both in inoculated and non-inoculated microcosms. Concomitantly, an accumulation of the two metabolites S-MET ethanesulfonic acid and S-MET oxanilic acid was found. Despite the dissipation of almost all the ATZ from the treated soils, the respective eluates were still highly toxic to an aquatic microalgae species, being as toxic as those from the untreated soil. We suggest that this high toxicity may be due to the S-MET and/or its metabolites remaining in the soil.Atrazine (ATZ) and  In particular, agricultural dealership sites and mix-load or disposal sites may represent potential sources of environmental contamination with high levels of diverse compounds 50 = 28–150 days, field, aerobic) and mobility  Pseudomonas sp. ADP is the best-characterized ATZ-mineralizing bacteria and uses ATZ as sole N source by means of a catabolic pathway encoded in the plasmid pADP-1 In soil, ATZ presents moderate persistency  and moderately mobile (KOC = 226 L kg−1) in soil, but very persistent in water S-enantiomer have the potential to cause adverse effects on microorganisms S-MET on ATZ-degradation in soil have been limited to indigenous soil microorganisms and intrinsic biodegradation. In some cases a negative effect was demonstrated S-MET S-MET could negatively affect the survival and the ATZ-biodegradation performance of the bioaugmentation bacterium Pseudomonas sp. strain ADP in a crop soil. We used a representative crop soil from Central Portugal S-MET . Doses mimicked worst-case scenarios of soil contamination, being thus higher than the recommended dose (RD) for weed control in corn plantations. The following issues were addressed: first, the ability of Pseudomonas sp. ADP, either combined or not with citrate amendment, to mineralize [ring-UL-14C]ATZ mixed with increasing doses of Primextra S-Gold (up to 50×RD), in soil at small laboratory scale. Second, the effects of S-MET in a pure form on the bacteria survival and on its ATZ-mineralizing ability, at concentrations of the pure active ingredients representing up to 50×RD, also in small soil microcosms. Third, the rate and extent of biodegradation of ATZ from 20× or 50×RD Primextra S-Gold when applying the bioremediation tool, in larger and more realistic soil microcosms Pseudokirchneriella subcapitata growth tests) on the eluates from soil samples. At this stage, eluates from soils contaminated with the herbicidal formulation Atrazerba FL (with ATZ as single active ingredient) and previously reported to be decontaminated after a 10-days treatment with the bioremediation tool S-MET initially present in the soil microcosms contaminated with Primextra S-Gold as well as the possible formation of its major degradation products S-MET oxanilic acid (OA) and S-MET ethanesulfonic acid (ESA) Possible deleterious effects exerted by 7 CFU g−1 soil dry weight) of soil spiked with mixtures of [14C]ATZ plus increasing doses of non-labeled ATZ from the double formulation Primextra S-Gold , ATZ mineralization started rapidly. The percentage of 14CO2 produced from the 14C-labeled ATZ attained maximal values within 3 days  from ∼1 (in the crop soil used) up to ∼50 14CO2 formation  and up to 60 µg g−1, did not significantly affect the bacterial survival  combined with soil amendment with trisodium citrate (Cs∶Natz∼50) −1, in only 5 days (−1 soil dry weight), inoculation with the bacterium without citrate amendment led to quite high levels of ATZ still remaining in the soil, namely 2.4 and 1.4±0.8 µg ATZ g−1 at days 5 and 8, respectively , at time zero, to 27±9 and 46±19 µg kg−1 after 5 days of incubation, in the 20×RD and the 50×RD-contaminated soils, respectively. The S-MET OA concentration increased from <6 to 42±22 and 87±33 µg kg−1, respectively, in the same period of time. At day 8, the concentrations of these compounds measured in the soil microcosms were somewhat lower, namely 22±2 and 28±7 µg S-MET ESA kg−1, or 29±5 and 43±35 µg S-MET OA kg−1, in the 20×RD and the 50×RD-contaminated soils, respectively (data not shown).Dissipation of ted soil . Indeed,at day 5 , and maiPseudomonas sp. ADP plus citrate  to soil, and to assess the efficacy of treatment of these soils with the bioremediation tool, the ecotoxicity to microalgae was assessed in eluates prepared from soil samples collected in the microcosms during the biodegradation experiments with  citrate , as examThe eluates from the soil microcosms that were spiked with Primextra S-Gold but not subjected to biaugmentation/biostimulation treatment caused serious deleterious effects on microalgae growth, compared with those from soil not contaminated with the commercial formulation . After tS-MET (and/or its degradation products) that remained in the soil during the biodegradation experiments in the microcosms contaminated with Primextra S-Gold (S-MET in the pure form (25 or 62 µg g−1 soil dry weight) were also highly toxic to the microalgae irrespective of the soil having been subjected or not to the Pseudomonas sp. ADP plus citrate treatment  of the formulation Atrazerba FL S-MET in the soil at concentrations between 15 and 50 µg g−1 of soil, representative of applications of up to approximately 50×RD of the double herbicidal formulation Primextra S-Gold, affected neither the survival of Pseudomonas sp. ADP used to bioaugment the contaminated soil (∼107 CFU g−1 of soil) nor the rate and extent of mineralization of 14C-labeled ATZ by this bacterium. Consistently, its growth curve in liquid PADP medium  was not affected as a result of medium supplementation with S-MET at concentrations up to 60 µg ml−1 . Based on the results obtained herein, we speculate that the observed moderate decrease in the bacterium ability to mineralize [14C]ATZ in the presence of increasing doses of Primextra S-Gold (from 5× up to 50×RD) may mainly reflect inhibitory effects on the bacterium physiology presumably caused by high concentrations of ATZ in soil  in a soil from a pesticide-contaminated site was not significantly affected by the presence of 50 µg g−1 of MET or of another herbicide, pendimethalin −1 of soil When considering Pseudomonas sp. ADP and citrate, ATZ removal from soil, due to its biodegradation, was extensive  and rapid (in less than 1 week) as reported before for the soils contaminated with the formulation Atrazerba FL that contains ATZ as the sole active herbicide P. subcapitata in the time-frame of the biodegradation experiments. Based on experimental evidences, we propose that the high ecotoxicity of these eluates may be mainly associated with the presence of S-MET that remained in the soil and/or of its degradates, as is further discussed below.Consistently, based solely on soil chemical analysis, we present evidences that in soil microcosms contaminated with 20× or 50×RD Primextra S-Gold and subsequently treated with optimized quantities of Pseudomonas sp. ADP apparently had no significant effect on the dissipation of S-MET in the soil microcosms contaminated with Primextra S-Gold, suggesting that this bacterial strain is not able to degrade the chloroacetanilide herbicide, as reported by others S-MET in the soils decreased moderately during the 8 days of the biodegradation experiments even though keeping values always higher than 60% of the initial concentration as described above. We thus suggest that the mobilization of a significant portion of intact S-MET from the soil microcosms to the water extracts may have contributed at least partially for the high ecotoxicity of the eluates towards the microalgae. Even though S-MET levels were not measured in the eluates prepared from the soil samples, using an estimated soil/solution distribution coefficient  approximately equal to 2.5 for S-MET in a sandy loam soil with 3.1% organic matter S-MET present in the soil may be mobilized into the water. Since a soil∶water 1∶10 ratio (w/v) was used in the eluate preparation, this points for predictable S-MET concentrations in the water extracts considerably higher (about 50-fold) than the value 8 µg L−1 reported as the acute 72 hours median effective concentration for P. subcapitata growth S-MET and other chloroacetanilide herbicides, being more susceptible than aquatic organisms from higher trophic levels S-MET were as highly inhibitors of microalgae growth as those prepared from the soils contaminated with Primextra S-Gold, the high ecotoxicity of the latter may be less likely attributable to the presence of ingredients from the commercial formulation other than S-MET . Nevertheless, possible toxic effects of unknown ingredients should not be ruled out as they might have been hidden due to the high toxicity exerted by S-MET (and/or derivatives).On one hand, soil bioaugmentation with S-MET ESA and S-MET OA did build-up in the soil microcosms concomitantly with the moderate dissipation of S-MET down to 60% of its initial value. MET can suffer biodegradation in soil EnterobacteriaceaThe metabolites S-MET ESA and OA that accumulated in the soils examined herein  able to concomitantly biodegrade multiple chemicals to less toxic derivatives; and, in the other hand, to address the effects of multiple pesticides on the performance of specific degrading microorganisms S-MET with the well-known ATZ-degrading bacterium Pseudomonas sp. ADP S-MET and other pesticides (including their possible metabolites) via water, mainly due to leaching and runoff events.In conclusion, the results herein presented point to the complexity of bioremediating soils contaminated with mixtures of pesticides, in this particular case of ATZ and S-metolachlor  and 4-morpholinepropanesulfonic acid (MOPS) were purchased from Sigma-Aldrich , trisodium citrate from Merck , and [ring-UL-14C]Atrazine  from American Radiolabeled Chemicals . The formulations Primextra S-Gold  for weeds in corn plantations) and Atrazerba FL  were purchased from Syngenta Portugal  and Sapec , respectively. The RD of ATZ and S-MET  were estimated as equivalent to approximately 0.8 and 1.0 µg g−1 of soil, respectively, assuming a possible field scenario of herbicide distribution through a 5 cm diameter ×10 cm height soil column and an average soil density of 1.5 g cm−3Atrazine , R) mutant of Pseudomonas sp. ADP which can mineralize atrazine with equal efficiency than the wild-type 8 colony forming units (CFU) ml−1, corresponding to OD640 nm∼1) grown at 30°C in liquid PADP medium (adapted from −1) as C source −1) from Atrazerba FL as sole N source g), and the cell pellet was washed once and resuspended in sterile saline solution (0.9% w/v NaCl). The concentrated cell suspension obtained was used as inoculum in survival, mineralization and biodegradation experiments, as is described below. The inoculum density, expressed as CFU ml−1, was determined by plating culture serial dilutions onto agarized Lennox Broth (LB) medium and further incubation at 30°C during 72 hours.A spontaneous rifampicin-resistant  was used A natural sandy loam soil  39.4±1.6%; cation exchange capacity 0.013 cmol gS-MET on the survival of Pseudomonas sp. ADP in ATZ-contaminated soil, experiments were carried out in sterilized EPA vials  each containing 5 g soil dry weight freshly spiked with ATZ to give an approximate concentration of 24 µg g−1 soil dry weight . Then, three different concentrations of S-MET  were added to these soils to represent doses equivalent to 15×, 30× and 60× higher than the RD of this active ingredient. Each compound was added from a stock solution in methanol , mixed with pure methanol when needed  and with sterile deionized water . Vials non-supplemented with S-MET were included as controls. Vials were vigorously stirred in a vortex to promote homogeneous distribution into the soil and were left uncapped in the laminar flow chamber for 1 hour to allow most of the methanol to evaporate. Each vial was then inoculated with an adequate amount of a suspension of bacteria viable cells prepared as described above, in order to have an initial inoculum density of approximately 107 CFU g−1 soil dry weight. Vials were stirred again, capped and incubated at 25.1±0.2°C in the dark. The total volume of liquid added to each vial  took in account the volumes of herbicide solutions, inoculum and deionized water. In each experiment, 16 replicates of each condition were prepared. To determine Pseudomonas sp. ADP RifR viable cells in soil, at each time interval (up to 9 days) one replicated vial was destructively sampled and processed immediately for the determination of CFU concentration as is described below . At least duplicate determinations from two or three independent experiments under identical conditions were carried out.To examine the effects of 14C]ATZ (stock solution in acetonitrile: 467.7 KBq ml−1) plus non-labeled ATZ was incorporated into the soil to give a total activity of 0.65 KBq g−1 dry weight of soil and different total concentrations of ATZ in soil. Non-labeled ATZ was supplied from Primextra S-Gold or as the pure substance, depending on the type of experiment to be carried out, as follows: first, to evaluate the effect of increasing doses of Primextra S-Gold in ATZ mineralization by Pseudomonas sp. ADP, soil in the vials was freshly spiked with mixtures containing the [ring-UL-14C]ATZ stock solution, aqueous suspensions of Primextra S-Gold  and sterile deionized water . Second, to examine the influence of S-MET in ATZ-mineralization bacterial activity, soil in the vials was freshly spiked with mixtures of the [ring-UL-14C]ATZ stock solution plus adequate amounts of stock solutions of herbicides in methanol to have total concentrations of 24 or 40 µg of ATZ g−1 soil dry weight plus, respectively, 30 or 50 µg g−1 of S-MET  or no added S-MET. Pure methanol  and sterile deionized water (to obtain soil moisture of 40% soil WHC) were also added. Then, soil was vigorously mixed with a vortex apparatus to incorporate the substances, followed by the inoculation with an adequate amount of the bacterial cell suspension prepared as described above (to obtain approximately 107 viable cells g−1 soil dry weight) s∶Natz equal to 50, as described before 14CO2. All vials were incubated at 25.1±0.2°C in the dark for up to 8 days. At adequate time intervals, the quantity of released 14CO2 was quantified using a Beckman LS 5000TD scintillation counter, as described before ATZ mineralization assays were carried out in sterilized EPA 40-ml vials containing 5 g soil dry weight, as previously described −1, respectively). After homogenization with a glass rod to promote incorporation of the herbicides, soil was inoculated with an adequate quantity of the inoculum bacterial suspension , and either amended or not with trisodium citrate at 1.3 or 3.4 mg g−1 soil dry weight in the 20× or 50×RD amended soils, respectively (to give a ratio Cs∶Natz∼50 S-MET (25 or 62 µg g−1 soil dry weight) instead of Primextra S-Gold, were also carried out for comparison purposes. Microcosms non-contaminated with the herbicides or contaminated but not inoculated with the bacterium were also included in each set of experiments as controls. In all experiments, soil moisture was adjusted to 40% soil WHC as described above, taking in account the total volume of liquid . Amended soils were again mixed, gently packed into the glass cylinders, and incubated at 25.1±0.2°C in the dark for up to 8 days. Soil microcosms were weighted every day to replace the water lost by evaporation with sterile deionized water. Soil samples were collected from the surface, at days 0, 1, 2, 5 and/or 8, and processed immediately for microbiology analysis (1.0±0.3 g dry weight), or stored at −20°C for chemical and ecotoxicological analyses (∼20 g), which were performed as described below. At least two determinations with samples from two independent experiments were carried out.Soil microcosms consisting of glass cylinders (10 cm height×4.5 cm interior diameter) containing 160 g dry weight of soil (∼7 cm×4.5 cm) over a 2 cm height layer of 2-mm-diameter glass beads supported by a fine Teflon mesh were used as described elsewhere Pseudokirchneriella subcapitata  were carried out on eluates obtained from soil samples collected in the biodegradation experiments  as described above. Microalgae culturing procedures were as previously outlined P. subcapitata growth tests followed standard guidelines −2 s−1). Three 900 µl sub-replicate cultures per replicated eluate, and a standard control with six replicates, were set up and inoculated with 100 µl of algal inoculum, so that the cell density at the start of the tests would be 104 cells ml−1. Both pH and conductivity were measured at the start of each test, and measured levels were comparable across treatments and not expected to have deleterious effects on the test organisms S-MET) on microalgae growth were evaluated through one-way analysis of variance followed by Dunnet's test to compare each dose with the respective non-contaminated control soil. One-tailed Student t-tests were employed to compare microalgae growth responses between spiked soils that were treated or not with the bioremediation tool, within the same herbicide dose and time interval. The latter tests were also used to assess decontamination efficacy due to bioaugmentation/biostimulation treatments, by comparing microalgae growth in eluates from non-contaminated (controls) and treated contaminated soils, within the same pesticide dose and time interval.Ecotoxicity tests with the model aquatic microalgae Pseudomonas sp. ADP RifR, soil samples from survival and biodegradation experiments were used as a basis for 10-fold dilution series in saline solution (0.9% NaCl w/v) in triplicate. Dilutions were spread plated onto agarized selective LB medium supplemented with rifampicin (50 mg L−1) and cycloheximide (100 mg L−1). Petri dishes were incubated at 30°C and colonies counted after 96 hours.To determine the concentration of CFU of −1 soil dry weight for ATZ, DEA, and DIA. For analysis of S-MET and of its OA and ESA derivatives, soil samples were dried at 40°C, extracted three times with a mixture of methanol and water (1∶2) using a Liarre 60 ultrasonic apparatus (15 min) and centrifuged for 15 min at 3000 g. Analysis of the combined extracts was performed by GC-EI-MS (Perkin Elmer-Clarus 500) for S-MET, and by LC-EI-MS (Agilent 1100 Series) for S-MET OA and S-MET ESA. All extracts were injected in scan mode to confirm the presence of each analyte and in SIM (single ion monitoring) for quantification purposes. Recovery ranged from 75 and 90% and the limit of quantification was 16 and 6 ng g−1 soil dry weight for S-MET and for S-MET ESA or S-MET OA, respectively.Soil samples were thawed at room temperature, dried at 40°C and further processed for analysis of the herbicides and respective metabolites in soil. For analysis of ATZ, DEA and DIA, extracts preparation and analysis by GC-Electron Ionization (EI)-MS (Perkin Elmer-Clarus 500) was carried out as described elsewhere


There are many reasons for dental implant failure, the development of bacteremia is concern for dentists. This is due to the possibility of unfavorable result such as implant loss or the need for re-treatment. In general, antibiotic prophylaxis is recommended for high risk patients such as individuals with an immunodeficiency, infectious endocarditis, or previous prosthetic instrumentation. However regarding clinically healthy, low and moderate risk individuals the use of antibiotics in implant dentistry is controversial. Another major concern regarding the over prescription of antibiotics is the selection of antibiotic resistant bacteria. A better understanding of the efficacy of prophylactic treatments regarding dental implantation is crucial. Thus a literature review and comparison of survival rates of dental implants with regimens of no, pre or post prophylaxis was performed using systematic literature review using reliable electronic databases. Retrospective or prospective controlled studies were examined for the influence of preoperative and/or postoperative or no antibiosis on dental implant success rate. Of the 11406 implants used in this literature review, cases with no antibiotics had a 92 % success rate, cases with pre-op antibiotic alone had a 96% success rate, cases with post-op antibiotic alone had a 97% success rate and cases with both pre and post-op antibiotic had a success rate of 96%. Thus, the results from this literature review show a > 90% success rate when antibiotics are used compared to when they are not used. Thus, no benefit is seen from the use of antibiotic prophylaxis in low and moderate risk dental implant patients.Dental implants; Pre-operative prophylactics; Post-operative prophylactics; Success rate Dentists and other physicians are often faced with the decision of whether to prescribe prophylactic antibiotics for complex oral surgeries such as dental implants. Although practitioners generally make these decisions on a case by case basis, if antibiotics were to be improperly prescribed it would produce a greater difficulty in treating legitimate infection . InteresAntibiotics are used to prevent infection if a dental implant does become infected the chances of failure are high . Though Early implant failure is commonly associated with certain strains of bacteria. The most common bacteria involved in these types of infections include: streptococci, anaerobic Gram positive cocci, and anaerobic gram negative rods . Thus, tThe American Heart Association recommends Amoxicillin and Penicillin as a first line of treatment due to their superior absorption and prolonged serum levels. However, in today’s population there is an increased level of penicillin allergies, thus a good alternative is clindamycin . The useRecent trends have explored the routine administration of antibiotic prophylaxis and look into the clinical research to substantiate its use. For decades, the use of antibiotics during dental surgical procedures has been condoned by major associations. According to the CDA, ”all dental procedures where significant oral bleeding and/or exposure to potentially contaminated tissue occurs typically (will) require antibiotic prophylaxis“ . The AmeThe pre- or postoperative uses of antibiotics in combination with dental implant surgery and success rates are poorly documented in the current literature. Pre-operational standard guidelines regarding antibiotics are possible to create; however, post-operational may remain open-ended and can be based on procedural outcomes during and after completion of the operation. Unfortunately, although multiple articles have been written, double blind control trials remain lacking because of ethics.The purpose of this paper is to review the current literature and information on dental implants and prophylaxis. Our objectives are to ask whether or not antibiotics are beneficial to implants, and in what instances pre- and/or postoperative antibiotic regimes should be prescribed. Hence we attempted to review the multitude of case series in literature to decipher this issue in clinical practice.The systematic literature review was completed using the electronic databases, Pubmed, Medpilot and Medline. Main search terms were antibiotic, prophyla combined with dental implant, implant failure, osseointegr and oral implant. Studies which met the inclusion criterion were English studies conducted between 1955 to January 2009 and which were retrospective or prospective controlled studies examining the influence of preoperative and/or postoperative or no antibiosis on dental implant failure rates. Administrations of various prophylactic antibiotics regimens were accepted. An unsuccessful dental implant was characterized by any implant which failed within the first 3 months, and studies with follow ups within the first 5 months were included. Studies with loading were not included and only studies using low risk patients were included. Of the 853 titles and abstracts which were identified by the literature search simply by word association, 797 were excluded due to either having no relevant material to implants and success rates regarding antibiotics, or were not prospective or retrospective studies. Fifty-six were selected for detailed review due to a relevant title. The next 45 titles were not acceptable because while relevant did not provide information on the antibiotic regimens prescribed, failed to provide a proper timeline of the implant procedure and/or were unclear or not specific enough to draw conclusive patient data. Among these, six met the criteria stated above and were included in our review .The results of the literature review showed no significant difference between the success rate of implants with and without the use of antibiotics. Implants performed with the use of antibiotics had a success rate of 96.5% while surgeries performed without antibiotics had a slightly lower success rate of 92%. When pre and post-op antibiotics were compared, success rates of 96% and 97% were found respectively . The oveThe literature search performed produced an equal division between studies supporting the use of antibiotic prophylaxis and those negating the use of antibiotics. However, a draw back in the current literature became evident as many studies were excluded from this literature review because they did not include comparisons between no antibiotic, pre-op, post-op and both pre and post-op antibiotic use. However, this was a significant finding on its own in that many more studies are required to help validate and improve current guidelines regarding antibiotic use and oral implants.One of the only commonalities amongst most of the papers was the feeling that antibiotics are overused and that the authors requested that all practitioners’ assess each patient individually in the hopes of reducing the amount of prescribed antibiotics. Of the papers which met the inclusion criterion, 50% agreed that pre- or post operational antibiotics were of benefit while the remaining 50% believed that antibiotics were not of any benefit. With such contrasting results one wonders how these studies could have differed so significantly. Differences may have been inherent within the patient population; healthier patients with stronger immunity versus patients with lower resistance, the number of times patients in studies had been previously prescribed antibiotics, cross-reactions with other drugs, or concomitant illnesses. Unfortunately, this reveals that much of the research completed to date on this topic is inconsistent and lacking in validity. For instance, no randomized controlled studies have been done. In addition, due to the lack of standardization many studies were difficult to compare and had to be excluded. Thus, until larger studies can be completed it will be difficult to determine a definitive answer.Some alternative methods of lowering the risk of infection that have been explored include the use of Chlorhexidine digluconate (CHX), a mouthwash rinse which is often used in conjunction with dental implants. CHX, when rinsed preoperatively has been proven to be an effective aid in promoting healing and reducing surgical complications . CHX alsAfter a thorough analysis of the literature, one can conclude that there is no clear evidence pointing to the need for prophylaxis antibiotics in conjunction with dental implant surgery. Of every million patients receiving just a single dose of oral amoxicillin, mild, moderate, and severe allergic reactions have been estimated to occur in 2400, 400, and 0.9 patients, respectively . The denThe scientific literature supports the limited use of prophylactic antibiotics, yet clinicians are continually over prescribing them . This noThe most common adverse effects of antibiotics are direct toxicity, hypersensitivity reactions, and the short or long term development of resistant microorganisms. Direct toxicity includes gastrointestinal , hematological concerns , alterations in the body’s normal flora leading to candidal infections or pseudomembranous colitis, nephrotoxicity , neuropathy , alterations in drug interactions, and finally hepatobiliary (jaundice or hepatitis) .Hypersensitivity reactions can range form mild to lethal. Mild reactions include cutaneous eruptions such as rashes, exfoliate dermatitis, or uticaria. Another complication with antibiotics is known as serum sickness, which is an immune complex condition. The most severe form is an immediate hypersensitivity including anaphylaxis, brochospasms, or laryngeal edema .From a long-term perspective one must be able to appreciate the concerns when a patient develops antibiotic resistant microorganisms. This is a potentially catastrophic concern, which is very difficult to measure. In addition, there is a tremendous financial concern with respect to the development of new drug therapies to treat such patients. The negative effects associated with use of antibiotic therapy must be assessed in comparison to the costs and morbidity related to treating infective endocarditis or infected prosthetic materials. If the risk-benefit and cost-benefit ratios are thoroughly assessed, it becomes clear that if there are specific therapeutic indications based on sound physiologic, anatomic and scientific evidence, then antibiotic prophylactic therapy may be justified . However


In situ endothelialization of cardiovascular implants has emerged in recent years as an attractive means of targeting the persistent problems of thrombosis and intimal hyperplasia. This study aimed to investigate the efficacy of immobilizing anti-CD34 antibodies onto a POSS-PCU nanocomposite polymer surface to sequester endothelial progenitor cells (EPCs) from human blood, and to characterize the surface properties and hemocompatibility of this surface. Amine-functionalized fumed silica was used to covalently conjugate anti-CD34 to the polymer surface. Water contact angle, fluorescence microscopy, and scanning electron microscopy were used for surface characterization. Peripheral blood mononuclear cells (PBMCs) were seeded on modified and pristine POSS-PCU polymer films. After 7 days, adhered cells were immunostained for the expression of EPC and endothelial cell markers, and assessed for the formation of EPC colonies. Hemocompatibility was assessed by thromboelastography, and platelet activation and adhesion assays. The number of EPC colonies formed on anti-CD34-coated POSS-PCU surfaces was not significantly higher than that of POSS-PCU . However, antibody conjugation significantly improved hemocompatibility, as seen from the prolonged reaction and clotting times, decreased angle and maximum amplitude (p<0.05), as well as decreased platelet adhesion  and activation. Here, we demonstrate that POSS-PCU surface immobilized anti-CD34 antibodies selectively captured CD34+ cells from peripheral blood, although only a minority of these were EPCs. Nevertheless, antibody conjugation significantly improves the hemocompatibility of POSS-PCU, and should therefore continue to be explored in combination with other strategies to improve the specificity of EPC capture to promote in situ endothelialization. With the rapid advancement of interventional cardiology over the past decade, percutaneous coronary intervention (PCI) has become the treatment of choice for atherosclerotic coronary artery disease (CAD) . AlthougDespite the clear short- to mid-term benefits of using DES over BMS, there have been concerns over the long-term safety of DES. Indeed, a recent meta-analysis in the Cochrane Review revealed that there were not statistically significant difference in death, acute myocardial infarction (MI), or thrombosis rates when comparing DES to BMS . FurtherA hemocompatible polymer stent coating is therefore a crucial element, as while it must act as a protective coating for the bare metal surface to prevent the problem of ISR as seen with BMS, it must not evoke an immunological hypersensitive response and subsequent stent thrombosis, which is the unaddressed problem currently seen with DES. This would carry with it the additional benefit of shortening or even doing away with the need for long-term dual anti-platelet therapy, which is currently the case with DES .in vivo compared to PTFE and PCU, evoking negligible immunoreactivity [We have developed and patented a proprietary nanocomposite polymer, polyhedral oligomeric silsesquioxane-poly(carbonate-urea) urethane (POSS-PCU), to meet the need for functional nanomaterials for biomedical applications. The introduction of inert nano-sized POSS moieties into PCU has been shown to greatly enhance the mechanical, physical, and thermal properties of PCU, such as tensile strength, viscoelasticity, chemical stability, and calcification resistance -20. Furtactivity ,20-23. Aactivity . Its supactivity , lacrimaactivity , vasculaactivity ,26, as wactivity , and is in situ endothelialization, which would then theoretically increase the probability of EPC capture and minimize the risk of VSMC proliferation. Ideally, stable integration of antibodies into POSS-PCU in the correct orientation for target cell-binding should be achieved without any significant detrimental effects on the physical properties and surface chemistry of POSS-PCU. To this end, de Mel (2011) investigated the possibility of incorporating amine-functionalized fumed silica (FS) into POSS-PCU to act as a nano-anchor for antibodies and peptides, and successfully demonstrated that the addition of amine-functionalized FS was able to facilitate antibody conjugation without negatively impacting the physical and biological properties of POSS-PCU [For POSS-PCU to be a favourable platform for EPCs to specifically adhere to and proliferate on, biofunctionalization of POSS-PCU through the incorporation of EPC-specific antibodies such as anti-CD34 is necessary to achieve POSS-PCU .2 groups in POSS-PCU-FS and the NH2 groups on the Fc region of antibodies  in a stable manner and in the correct orientation, EDC-NHS (N-ethyl-N’-(3-dimethylaminopropyl)-carbodiimide, N-hydroxy-succinimide) was chosen as a chemical linker between the NHtibodies . This isin vitro study aimed to characterise the anti-CD34 biofunctionalized POSS-PCU surface, and assess its ability to both enhance EPC capture from peripheral blood as well as increase hemocompatibility.As such, this All chemical reagents were purchased from Sigma-Aldrich UK, and cell culture reagents were purchased from Life Technologies (Invitrogen) UK, unless otherwise stated. All experiments were conducted in triplicates (n=3) unless otherwise stated.DMAc) was subsequently added slowly to dissolve the pre-polymer, forming a solution that was then cooled to 40°C. Following solvation, chain extension of the pre-polymer solution was achieved by drop-wise addition of a mixture of ethylenediamine and diethylamine (in DMAc) to form a final polymer solution of POSS-modified polycarbonate urea-urethane (in DMAc).The synthesis of our proprietary POSS-PCU has been described in detail elsewhere . Briefly2) groups was carried out as follows. 1g of A200 fumed silica was first mixed with 100 mL of propan-2-ol, 5mL of 3-aminopropyltriethoxysilane (APTES), and 5mL of deionised water in a pre-silanised round-bottomed flask. This mixture was sonicated in a bath sonicator  for 45min. To prevent overheating, the flask containing the mixture was cooled in ice, and the water in the sonicator was changed every 15min. Subsequently, the resultant mixture was refluxed at 70 °C for 6hrs. Following which, the mixture was centrifugated at 4000rpm for 40min. The resultant supernatant was discarded, while the pellet was re-suspended in propan-2-ol and centrifugated again. This step was repeated 5 times. Finally, the pellet was placed in a heating oven (Binder GmbH) at 65 °C for 24hrs. The resultant amine-functionalized fumed silica (FS-NH2) had a powdery, white appearance.In-depth discussion about amine-functionalization of fumed silica was developed in our lab by de Mel, and is described elsewhere . Briefly2 incorporation was developed in our lab by de Mel, and is described elsewhere [2 nanoparticles were incorporated into POSS-PCU (18% in DMAc) to make up a POSS-PCU-FS mixture (2% FS-NH2 in 18% POSS-PCU) as follows. First, FS-NH2 was added to DMAc in a glass scintillation vial, and dissolving by sonication for 30min at room temperature in a bath sonicator. The dissolved FS-NH2 was then added to 18% POSS-PCU and subjected to further mixing with an Ultrasonic Processor  for three 20s cycles. The mixture was left to stand for an hour in order to allow air bubbles to dissolve.FS-NHlsewhere . Briefly2 surface area) using a cutting press and die , in order to fit 24-well plates. The discs were then autoclaved before antibody conjugation where necessary. Preliminary studies have shown that autoclaving does not negatively affect antibody conjugation to polymer (data not shown).Thin POSS-PCU and POSS-PCU-FS polymer sheets were made by casting POSS-PCU and POSS-PCU-FS polymer mixtures on separate 10cm by 10cm stainless steel plates and leaving them to dry in a heating oven 65°C for 24hrs. Following which, polymer sheets were peeled off and cut into discs . Subsequently, the discs were washed with PBS to remove any unreacted functionalities.2 for a 2cm2 polymer disc. The diluted antibody solutions were then filter-sterilised in a laminar flow hood. Either anti-CD34 or anti-IgG antibody dilutions were added to their respective EDC-NHS-activated POSS-PCU-FS discs, and placed on the shaker for 30min in the dark at room temperature. The discs were then transferred to a 4°C fridge and incubated overnight in the dark to facilitate covalent immobilization of antibodies. Subsequently, discs were washed with PBS to remove non-specifically adsorbed antibodies.Mouse anti-human CD34 and mouse anti-human IgG antibodies were both separately diluted in 0.1% PBS-Tween 20 (PBST) to a final concentration of 2µg/mL, which gives a surface coating density of 1µg/cmSurface morphology of POSS-PCU, POSS-PCU-FS, and anti-CD34-coated POSS-PCU-FS (POSS-PCU-FS+CD34) was assessed using scanning electron microscopy (SEM) . Samples were first given 20nm gold coating in a Quroum Q150T Sputter Coater using argon prior to SEM imaging.Evaluation of the presence of immobilized anti-CD34 as well as the uniformity and stability of this antibody coating over time was assessed by immunostaining with Quantum Dot (QD)-anti-IgG conjugates. POSS-PCU-FS+CD34 discs were first washed in deionised water, then immersed in PBS and left on the shaker at room temperature in the dark. After 24hrs and 72hrs, discs were removed and incubated with red QD-anti-IgG (goat anti-mouse) conjugates for detection of immobilized mouse anti-CD34. Fluorescent micrographs were taken, and the fluorescence intensities of QD-anti-IgG immunostained POSS-PCU-FS+CD34 surfaces after 24 and 72hrs (n=3 per time point) were quantified using ImageJ (NIH) software. Unmodified POSS-PCU and POSS-PCU-FS were used as controls.Water contact angle (WCA) measurements were performed for POSS-PCU, POSS-PCU-FS, and POSS-PCU-FS+CD34 films with a KRÜSS DSA 100 machine and Drop Shape Analysis software  to compare the differences in surface wettability. The sessile drop method was used, with droplet volume and dispensing rate kept constant at 5.0µl and 195.1µl/min respectively. A total of 6 measurements were taken randomly at different spots of each disc (n=6), each within 10s of dispensing. Surfaces with WCA measurements of > 90° are considered hydrophobic, whereas those with angles < 90° are considered hydrophilic.All procedures that involved extraction of human blood via venipuncture were obtained from healthy volunteers, with informed consent, and approved by the Institutional Review Board (IRB) at the Division of Surgery and Interventional Science at University College London and Royal Free London NHS Foundation Trust.20mL blood samples were collected with informed consent ) from healthy adult human volunteers by venepuncture in ethylenediaminetetraacetic acid (EDTA) BD Vacutainer®. Samples were thoroughly mixed with the EDTA to prevent coagulation.g for 30min at room temperature. The buffy coat layer containing the mononuclear fraction of peripheral blood, including EPCs, was carefully harvested and washed with Hank’s Balanced Salt Solution (HBSS) by centrifuging at 250g for 10min, discarding the supernatant, and re-suspending the pellet in HBSS. After 2 rounds of washing, isolated PBMCs were re-suspended in cell culture media (CCM), which comprised Medium 199 supplemented with 20% FBS, 1% glutamine, and 1% penicillin/streptomycin . PBMCs were then enumerated using a hemocytometer and Trypan Blue exclusion dye, and then diluted in CCM to a final concentration of 1.0x106 PBMCs/mL.EPC isolation was carried out within an hour of blood sample collection, using a method that has been reported previously to produce EPC colonies that are able to differentiate into cells with EC markers and characteristics . First, 6 PBMCs/well were seeded in 6-well plates. After 3 days of culture, non-adherent cells were removed by gentle washing with PBS, while adherent cells were kept in culture for further cultivation. On days 5 and 7 post-seeding, half of the media in each well was carefully replaced with fresh CCM. The cell cultures were visualised every 2-3 days under light microscopy, and any eventful changes in cell morphology were recorded. After 7 days, cells were fixed and directly immunostained with QD-antibody conjugates, for the expression of CD34, VEGFR-2, CD31 , and von Willebrand factor (vWF).For EPC characterization, 2–3 x 10oC/5% CO2 incubator overnight. The next day, the CCM in each well was replaced by 1mL of 1x106 PBMCs/mL PBMC suspension, and the plate was returned to the 37 oC/5% CO2 incubator. For positive controls, PBMCs were seeded in empty wells, whereas POSS-PCU-FS discs with no cells seeded were used as negative controls. After 3 days of culture, non-adherent cells were removed by gentle washing with PBS, while adherent cells were kept in culture for further cultivation. On days 5 and 7 post-seeding, half of the media in each well was carefully replaced with fresh CCM. At day 7, cells were fixed and double-stained for EPC markers (CD34 and VEGFR-2) as well as EC markers (CD31 and vWF). In addition, the total number of EPC colonies formed per 106 PBMCs seeded in each well was enumerated manually. EPC colonies were defined morphologically as central clusters of rounded cells with spindle-shaped cells emanating from the centre, which also stained positive for both CD34 and VEGFR-2 under a laser scanning confocal microscope [For the EPC capture assay, sterile anti-CD34-coated POSS-PCU-FS, mouse IgG isotype-coated POSS-PCU-FS (negative control), as well as unmodified POSS-PCU and POSS-PCU-FS discs, were placed in a 24-well plate and equilibrated in sterile CCM in a 37 croscope -34.Prior to direct immunostaining with QD-antibody conjugates, cells cultured on the various surfaces were first washed twice with 0.1% PBST, fixed with 2% paraformaldehyde  for 20min, then washed thrice with 0.1% PBST at room temperature. Cells were subsequently permeabilized in 0.5% Tween-20 (in PBS) for 15min, then washed thrice with 0.1% PBST.The fixed cells were first blocked with 1% bovine serum albumin  for ½hr at 4°C in the dark, and then washed with 0.1% PBST before incubation with QD-antibody conjugates for 2hrs at 4°C in the dark. Samples were subsequently washed with 0.1% PBST, incubated with 4',6-diamidino-2-phenylindole  for 5min at room temperature, washed with 0.1% PBST, and left in 0.1% PBST for imaging. Images were captured by a Nikon Eclipse TE300 inverted microscope equipped with an EZ-C1 confocal microscopy system (Nikon). A 488nm He-Ne excitation laser was used, and images were acquired using either a 515-530nm emission range channel (for green and blue fluorescence detection), or a 650LP channel (for red fluorescence detection).Anti-CD34 (241µg/mL), anti-VEGFR-2 (250µg/mL), anti-vWF , and anti-CD31 (500µg/mL) antibodies were conjugated with either red or green quantum dots and diluted in 1% BSA to make up 0.25µg/mL QD-antibody working solutions. VEGFR-2 and CD34 are EPC markers, whereas vWF and CD31 are EC markers. Hence, the following pairings of QD-antibodies were used: QD-anti-VEGFR-2  and QD-anti-CD34 ; QD-anti-vWF (red) and anti-CD31 (green). Additionally, cells were stained with blue DAPI nuclei counterstain.Thrombelastography (TEG) was carried out using the TEG coagulation analyzer (TEG 5000® Thrombelastograph Haemostasis System) to evaluate contact activation and blood clotting kinetics of the anti-CD34-coated POSS-PCU-FS surface. TEG continuously monitors the entire clotting process and generates parameters that relate to each phase.2 in 18% POSS-PCU) and incubated upside down to dry in a 65°C oven overnight. These POSS-PCU-FS-coated cups were then incubated with EDC-NHS for 30min, washed with PBS, then incubated with anti-CD34 antibody solution (2µg/mL) overnight at 4°C. Anti-CD34 coated TEG cups were air-dried in a laminar flow hood prior to TEG analysis.Standard TEG cups were coated with a thin, uniform layer of POSS-PCU-FS polymer  were used as positive controls, whereas standard cups containing citrated blood (300µL) mixed with 0.01M L-arginine (40µL) were used as negative controls, as L-arginine is known to have anti-coagulant effects on native whole blood . To reduoC/5% CO2 incubator. For the isolation of platelet-rich plasma (PRP) by density centrifugation, a 20mL blood sample was first collected from a healthy volunteer using BD Vacutainer® containing 0.109M buffered sodium citrate, and mixed gently. The citrated blood was carefully layered onto 20mL of Histopaque-1077 and centrifuged at 200g for 30min. The topmost clear yellow layer making up the PRP was carefully harvested. Average platelet number was determined by making 3 reference counts with a hemocytometer, following which, the harvested PRP was diluted in sterile PBS to give a final working concentration of 1x106 platelets/mL.Sterile POSS-PCU, POSS-PCU-FS, and anti-CD34 coated POSS-PCU-FS discs were equilibrated overnight in sterile PBS in a 24-well plate placed in a 37 6 platelets/mL), and incubated for 1 hour in a 37°C/5% CO2 incubator. After 1 hour, the supernatant in each well was gently transferred to separate Eppendorf tubes, and the number of platelets enumerated immediately using a hemocytometer.PBS in each well was replaced with 1mL of diluted PRP (1x10The degree of platelet adhesion to each sample was expressed quantitatively as a Platelet Adhesion Index (PAI), which was calculated as follows:i is the initial number of platelets seeded on each sample (106), and Ps is the number remaining in the supernatant after the 1 hour incubation. A staging for platelet activation was previously described by Ko and Cooper (1993) [where Pr (1993) , and is For morphometric analysis of the surface-activated platelets, the polymer discs were retained following removal of the supernatant, and prepared for SEM. This was done by gently washing the discs with PBS, and then fixing any adhered platelets with 4% PFA for 20min, followed by dehydration with ascending concentrations of ethanol , with 20min of incubation for each step. This method of fixation and dehydration protects platelets from desiccation while concurrently reducing unwanted artefacts, thereby preserving platelet integrity for high resolution morphological analysis under SEM. The samples were air-dried in the laminar flow hood for 16–24hrs prior to SEM imaging.p <0.05 was considered statistically significant.Parametric data has been expressed as mean ± standard deviation. SPSS 21.0 (IBM) was used to perform one-way ANOVA with post-hoc Bonferonni’s test for analysis of statistical significance. POSS-PCU displayed a rough and textured surface . This isp < 0.05) (POSS-PCU had a water contact angle of 110.0° ± 4.1°. POSS-PCU-FS had a slightly reduced water contact angle of 103.1° ± 3.2°. Conjugation with anti-CD34 antibodies resulted in a decreased water contact angle of 80.0° ± 4.5°( < 0.05) .p > 0.05) (Immunostaining using red QD-anti-mouse IgG (secondary antibody) confirmed the presence of anti-CD34 antibodies (primary antibody) on the polymer surface even after 24 and 72 hours of continuous washing. Fluorescence imaging revealed a uniform distribution of anti-CD34 antibodies. Results indicated the stability of antibody coating, without significant reduction in fluorescence intensity after 72 hours  .7 ± 2.94 × 106 PBMCs were isolated from 20 ml of venous blood in with a protocol that was described previously [6.32 × 10eviously ,28,31. Tp < 0.05) (p > 0.05). EPC colony numbers on positive control (10.3 ± 2.1) was higher and statistically significant compared to all other groups (p < 0.05). DAPI staining revealed that POSS-PCU-FS and POSS-PCU-FS+CD34 had a higher density of adhered cell clusters compared to POSS-PCU and POSS-PCU-FS+IgG. This high density of cell clusters appeared to be similar to positive control. Immunostaining for EPC markers showed that a small clusters of POSS-PCU-FS+CD34 were double-positive for CD34+/VEGFR-2+  was monitored over a period of 7 days. Colonies were identified by QD-immunostaining for EPC markers (CD34/VEGFR-2) . The abi < 0.05) . No statVEGFR-2+ . None ofTEG was used to assess the degree of contact activation of the coagulation cascade on the various surfaces of polymer samples. All test groups demonstrated a typical cigar-shaped TEG profile, indicating functional coagulation in normal hemostasis .r-time, k-time, α-angle, MA). POSS-PCU-FS+CD34 had an r-time of 21.3 ± 3.5. This was statistically significant (p < 0.05) from positive control, POSS-PCU, and POSS-PCU-FS, which had r-time of 9.0 ± 0.5, 5.6 ± 2.6, and 10.9 ± 2.6 respectively. r-time for POSS-PCU-FS+CD34 was not statistically significant (p > 0.05) compared to negative control (22.6 ± 2.4). POSS-PCU-FS+CD34 had a k-time of 20.0 ± 0.0. This was statistically significant (p < 0.05) from positive control, POSS-PCU, POSS-PCU-FS, and negative control which had k-time of 2.7 ± 0.3, 2.2 ± 0.3, 8.3 ± 0.5, and 7.8 ± 0.3 respectively. The α-angle for POSS-PCU-FS-CD34 was 10.4 ± 5.1. This was statistically significant (p < 0.05) compared to positive control, POSS-PCU, POSS-PCU-FS, and negative control, which had α-angles of 53.3 ± 3.4, 60.9 ± 3.6, 24.3 ± 3.0, and 32.7 ± 2.5 respectively. The MA for POSS-PCU-FS+CD34 was 10.5 ± 1.0. This was statistically significant from positive control, POSS-PCU, POSS-PCU-FS, and negative control, which had MA values of 65.0 ± 1.0, 60.6 ± 2.6, 42.1 ± 1.5, and 52.3 ± 2.1 respectively  (p < 0.05). Immobilization of anti-CD34 antibodies further reduced the PAI to 8.4 ± 0.7 (p < 0.05).Results of the platelet adhesion assay largely agrees with TEG and platelet activation assay. A high density of platelet adhesion on POSS-PCU was observed, which was corroborated by a high Platelet Adhesion Index (PAI), which was almost twice that seen in collagen  . PAI of The main premise of developing a biofuntionalized polymer for cardiovascular applications is to circumvent restenosis and thrombosis in order to sustain long-term patency and viability. Furthermore, adopting a “pro-healing” approach such as mobilizing endothelial progenitor cells to form a confluent layer of endothelium is one way to restore vessel homeostasis. POSS-PCU has been previously shown to function as a viable material for cardiovascular applications due to its superior biocompatibility and mechanical properties ,38. ThisThe native vascular endothelium normally provides an efficient anti-thrombotic and anti-coagulant barrier against thrombus formation, lipid uptake, and inflammatory cell migration . It alsoin vitro, before implanting the autologous endothelium back into the patient [in vitro endothelialization is time-consuming and expensive, with a high risk of contamination [in situ [Early studies focussed on cultivating patient-isolated ECs to confluence on implant surfaces  patient -41. Howemination ,42. An a[in situ ,29.in vitro [EPCs are bone marrow-derived stem cells, which were first isolated from peripheral blood by Asahara et al. (1997) and shown to attain EC characteristics in vitro . Althougin vitro ,45. Addiin vitro ,32,46,47in vitro; thirdly, their ability to retain a capacity for differentiation; and fourthly, their ability to migrate to sites of vascular injury. Numerous in vitro and in vivo studies have successfully demonstrated induction of EPC mobilisation, homing, incorporation to target sites, and/or differentiation into an EC monolayer. Notably, a substantial number of these studies employed surfaces biofunctionalized with the EPC-specific anti-CD34 antibody to capture circulating EPCs [EPCs have been widely investigated for tissue engineering applications for a number of reasons: firstly, their relative ease of isolation; secondly, their high capacity for proliferation ing EPCs ,28,48-54TM Stent (GS) (OrbusNeich), which follow-up studies have shown to be safe even after 3 years post-implantation [in vitro and in vivo experiments by Larsen et al. (2012) provide further support for anti-CD34 immobilization as a viable strategy for EPC capture [in vitro EPC-capture assay demonstrated a preferential adhesion of human peripheral blood-derived CD34+ cells to GS over BMS. An in vivo baboon AV shunt model showed that GS inhibits in-stent thrombosis, as indicated by a significantly lowered platelet deposition compared to BMS, while in a rabbit endothelial denudation model, analysis of EC markers expression after 7 days indicated that GS significantly promotes re-endothelialization [Encouraging results have been achieved with the anti-CD34-coated Genousantation ,52,53,55antation . Anti-CDin situ.Despite the large amount of literature on the EPC capture efficacy of anti-CD34-coated surfaces, much less work has been done on the hemocompatibility of these modified surfaces. While these surfaces aim to increase EPC adhesion, they inadvertently run the risk of increasing clotting tendencies by encouraging non-specific platelet or leukocyte adhesion . It is tImmobilization of anti-CD34 antibodies via amine-functionalized FS and an EDC-NHS covalent linker proved to be a feasible method for obtaining a functional and stable surface for EPC capture. Distinct surface modifications were observed via SEM imaging, and a reduction of water contact angle was also seen after antibody immobilization.+ cells via a specific binding, rather than a non-specific binding. However, the majority of cells captured on POSS-PCU-FS+CD34 were single-positive, being CD34+/VEGFR-2- or CD34-/VEGFR-2-. The number of EPC colonies formed on POSS-PCU-FS+CD34 was significantly higher than POSS-PCU-FS-IgG (p < 0.05), but not POSS-PCU and POSS-PCU-FS (p > 0.05).Extensive cell adhesion and clustering was observed on POSS-PCU-FS+CD34 and POSS-PCU-FS after 7 days of culture, compared to POSS-PCU and POSS-PCU-FS+IgG. The higher degree of cell adhesion on POSS-PCU-FS compared to POSS-PCU could perhaps be explained by the positively charged amines on the surface of POSS-PCU-FS. The majority of the cells on POSS-PCU-FS+CD34 were CD34 positive, while none of the cells on non-specific IgG isotype negative control (POSS-PCU-FS+CD34) were CD34 positive. This indicates that immobilized anti-CD34 antibodies on POSS-PCU-FS+CD34 capture CD34+ cells have the potential to differentiate into mature EC and VSMCs. Furthermore, EPCs can release growth factors such as VEGF, which can upregulate mobilization and proliferation of VSMCs [Literature on in vitro and in vivo studies indicate that CD34of VSMCs ,42. Anotof VSMCs . Evidencof VSMCs . Howeverof VSMCs . Hence, of VSMCs ,29,30,52The inability of adhered EPCs differentiating into ECs could be explained by the relatively short culture period of 7 days. The static culture was not representative of cells within the luminal area of blood vessels, which are in 3D and exposed to flow conditions. Evidence suggests that cells grown in 3D perfused reactors might be more representative of physiological conditions compared to 2D static conditions. Furthermore, it has been shown that fluid shear stress can influence the differentiation of progenitor cells into endothelial cells . TherefoTo ascertain the blood-compatibility of POSS-PCU and its antibody-immobilized version, contact activation and platelet assessments were carried out. TEG is used to assess coagulation kinetics in real-time, and is used in cardiac and transplant surgery. Therefore, TEG cuvettes were coated with the various test samples with the intention of seeing how blood reacts with them compared to uncoated cuvettes. POSS-PCU was observed to have the lowest reaction time (r-time: 5.6 ± 2.6 min) and clotting time (k-time: 2.2 ± 0.3 min) compared to POSS-PCU-FS and POSS-PCU-FS+CD34. This could be due to its hydrophobicity, reflected in its high water contact angle (110.0 ± 4.1). The relationship between hydrophobicity and thrombogenicity, however, is contentious. While there is evidence suggesting that hydrophobic materials such as ePTFE are thrombogenic due to its affinity for plasma proteins such as fibrinogen , others p < 0.05) and reaction time  compared to POSS-PCU. This may be due to its reduced water contact angle . Further surface characterization studies would be necessary to confirm this relationship. Upon immobilization with anti-CD34 antibodies, resulted in increased reaction time  and increased clotting time , when compared to POSS-PCU-FS. This apparent reduction in thrombogenicity and improved hemocompatibility may be due to modifications in surface biochemistry and nano-architecture. Three-dimensional globular structures can be seen on POSS-PCU-FS+CD34. This might contribute to surface passivation via steric repulsion of platelets and clotting factors [POSS-PCU-FS displayed a longer clotting time , followed by POSS-PCU-FS (24.3 ± 3.0), and POSS-PCU-FS+CD34 (10.4 ± 5.1). MA is associated with the strength of clot, and indicates platelet-fibrin interactions. Similarly, POSS-PCU had the highest MA value (60.6 ± 2.6), followed by POSS-PCU-FS (24.1 ± 1.5), and POSS-PCU-FS+CD34 (10.5 ± 1.0).Results of platelet activation and adhesion largely correlated with TEG profiles. It was interesting to note that POSS-PCU demonstrated the highest degree of platelet adhesion, even exceeding that of collagen. Although showing a relatively high degree of adhesion, SEM revealed that platelets were largely inactivated on POSS-PCU. Indeed, there is complex relationship between platelets and mobilization of EPCs, and evidence has shown that a certain degree of platelet adhesion and activation is needed for EPC growth and proliferation . Hence, in vitro engineering mechanics, such as radial strength, percentrage recoil, and accelerated fatigue studies. In vivo studies would also have to be performed in animal models, and various studies like optical coherence tomography (OCT) to assess stent malapposition and the degree of re-endothelialization.Our future work regarding this aspect of biofunctionalized stent coatings would be to assess +, their potential to differentiate into functional endothelial cells remain open to question. Nevertheless, this approach of actively attracting EPCs for in situ endothelialization on POSS-PCU increased its hemocompatiblity. It might also be useful to consider using various bioactive moieties, e.g. VEGFR-2, to be used in tandem with anti-CD34 for capturing EPCs as this will increase its specificity. It is also pertinent to accurately characterize the identity and functionality of the captured EPCs on the biomaterial surface, as this would be an imperative design consideration for its long-term viability. Taken together, this avenue of promoting in situ endothelialization and hemocompatibility warrants further refinement as it is of relevance in a plethora of cardiovascular applications.We have demonstrated that clinical-grade POSS-PCU can be further bio-functionalized by attaching anti-CD34 antibodies on its surface. Anti-CD34 antibodies attached on POSS-PCU displayed increased cell adhesion, although it is likely that only a small sub-population of adhered cells were EPCs. It could be assumed that, although the vast majority of adhered cells were CD34


Dear Editor,The most prevalent complication of cholelithiasis is chronic cholecystitis usually necessitating cholecystectomy . The bilIn this cross sectional study, 132 patients with cholelithiasis and chronic cholecystitis without risk factors for postoperative sepsis were subjected to elective laparoscopic cholecystectomy from September 2009 to September 2010. We ordered antibiotic prophylaxy routinely with 1gr keflin one house before operation. Using a questionnaire, patients' age, sex, clinical features of the patients, the isolated bacteria, and antibiogeram were recorded. During surgery 5-10 ml of the bile samples were aspirated with sterile syringe from gallbladder immediately after cholecystectomy; 3-5 ml was placed in a sterile container; another 3-5 ml was inoculated directly into an aerobic and anaerobic BacT/Alert blood culture bottle. The Samples were sent immediately to the Laboratory and incubated at 37°C for 24h. After the bacteria were isolated, we performed antibiotic sensitivity tests by the isolates carried out using Kirby-Bauer method with a colony in Mueller-Hinton agar medium (pH 7.2-7.4). The results were reported as susceptible, resistant and intermediate based on the diameter of the clear zone around disks with reference to the antibiotic standard table. The Data were analyzed by Chi-Square test and P<0.05 was considered significant.B. fragilis and B. fusiformis may produce an E. coli substance called β-Lactamase which resolves bilirubin. The bilirubin when integrated with calcium ion forms calcium bilirubinate.From the total 132 bile samples, bacteria were isolated in 50 samples 37.87%). Some other studies have shown that 20-50% of the patients with chronic cholecystitis have positive bile culture . Al Harb.87%. SomThe difference in anaerobic and Polymicrobial positive culture between our study and others' could be attributed to the method of antibiotic therapy; here, we ordered antibiotic prophylaxy routinely while in Ballal et al.'s study it was selectively and in Al Harbi et al.'s study we had not known.E. coli was the most common isolate , as previously reported , Klebsiella pneumoniae  and Proteus . The significance of E. coli dominance is also supported by Maki's studies (1966) indicating a potential role for E. coli's Glucosonidase enzymatic activity in formation of calcium bilirubinate gall stone (E. coli (25%) as the most prevalent isolated bacterium, and the prevalence of Klebsiella, Aerobacter, Pseudomonas, Enterococci, and Proteus as 12%, 10%, 9%, 8%, and 3% respectively (B. fragilis (58.82%) predominant among the anaerobes (E. coli (26.1%), Enterococcus facials (15.6%), and Pseudomonas aeroginosa (9.6%) (E. coli was 92.3% susceptible to Amikacin and Enterococci 69.23% susceptible to Cephtriaxone. The most susceptible antibiotic for microorganisms on the whole was Amikacin. Ballal et al. have shown anaerobes were sensitive to Cefotaxime, Metronidazole, Chloramphenicol, Cefazolin, and tetracycline, and aerobes isolated to Ampicillin, Chloramphenicol, streptomycin, tetracycline, Gentamicin, and second generation Fluoroquinolones such as ciprofloxacin and Norfloxacin (In our study reported . Enterobll stone . In our ll stone , perhapsate 13; 2%, as preoccus 6; %, KlebsiE. coli 2% as the In spite of the fact that we ordered antibiotic prophylaxy for all of the patients, bacterial isolates and infectious complications showed no significant difference. Thus, we recommend starting antibiotics selectively, if supported by the clinical conditions and/or culture reports, in case of cholecystitis and cholelithiasis. However, routine culture of all bile samples is strongly advised.Given our and others' studies anaerobes are rare in the human biliary system; therefore, if antibiotic therapy is considered, aerobic coverage should be satisfactory. In addition because the most susceptible antibiotic for microorganisms on the whole was Amikacin, this antibiotic can be started selectively.


Sir,We read with great interest the editorial on the new metallo-β-lactamaseWhile several metallo-β-lactamases have been named after cities where they originated or were detected first without deleterious effects, the naming of the new one with our nation’s capital has had much negative impact21The objection to naming the plasmid after India’s capital actually arose after its misuse for discouraging medical tourism in India which is an economically growing enterpriseWe also propose that hereafter names of places or persons be avoided for newly discovered biological agents.Has the name helped in increasing public perception of the “disorganized state of antibiotic therapy” in India? The answer is no, but on the other hand the media and even government officials went into a defensive mode, thus missing a good opportunity to publicly discuss the need for organization and discipline of antibiotic availability, choice and clinical use so as to reduce the spread of existing antibiotic-resistant microbes and to prevent others from developing. We know that D. Ragunath is a leader in the area of antimicrobial resistance. We urge him, the Indian Council for Medical Research, the Departments of Health Research and Health & Family welfare and other concerned agencies to take the combat against antibiotic misuse on a ‘war footing’ with enforceable regulations and legislations.


The economic downturn exacerbates the inadequacy of resources for combating the worldwide HIV/AIDS pandemic and amplifies the need to improve the efficiency of HIV/AIDS programs.We used data envelopment analysis (DEA) to evaluate efficiency of national HIV/AIDS programs in transforming funding into services and implemented a Tobit model to identify determinants of the efficiency in 68 low- and middle-income countries. We considered the change from the lowest quartile to the average value of a variable a "notable" increase.Overall, the average efficiency in implementing HIV/AIDS programs was moderate (49.8%). Program efficiency varied enormously among countries with means by quartile of efficiency of 13.0%, 36.4%, 54.4% and 96.5%. A country's governance, financing mechanisms, and economic and demographic characteristics influence the program efficiency. For example, if countries achieved a notable increase in "voice and accountability" , the efficiency of their HIV/AIDS programs would increase by 40.8%. For countries in the lowest quartile of per capita gross national income (GNI), a notable increase in per capita GNI would increase the efficiency of AIDS programs by 45.0%.There may be substantial opportunity for improving the efficiency of AIDS services, by providing more services with existing resources. Actions beyond the health sector could be important factors affecting HIV/AIDS service delivery. Since the beginning of the epidemic of Human Immunodeficiency Virus/Acquired Immune Deficiency Syndrome (HIV/AIDS), almost 60 million people have been infected with HIV and 25 million people have died of HIV-related causes . DespiteEfficiency studies conducted at the organizational level  show a substantial variation of performance (measured by program unit cost) of HIV/AIDS interventions both within a country and across countries -9. WhileThis study uses comprehensive data released in 2008 by the Joint United Nations Programme on HIV/AIDS (UNAIDS)  to quantThis study followed a classical framework of economic analysis of efficiency using a two-step process: in the first step efficiency of national HIV/AIDS programs were evaluated with direct inputs and outputs, and in the second step econometric models were applied to explain the efficiency . Thus thWe used the data from UNAIDS reports on national AIDS spending to determine the inputs for national HIV/AIDS programs ,15. We sFour aspects have been highlighted by the World Bank to improve the efficiency of funding, which include 1) macroeconomic status, 2) social and cultural factors, 3) infrastructure and human resources, and 4) institutional and policy environment [Data on national HIV/AIDS expenditure and the three types of HIV/AIDS services  were obtained from UNAIDS documents ,16-19. AWe used output-oriented data envelopment analysis (DEA), a classic non-parametric approach to evaluating technical efficiency , to estiWe used DEA-solver 5.0 (Saitech Inc. New Jersey) to estimate two sets of efficiency scores for each observation using DEA models. The first set was obtained by running the DEA model on separate years (each country was compared to its peers in the same year) to estimate the efficiency of countries in implementing HIV/AIDS programs (hereafter termed 'separate DEA'). The second set was calculated by pooling all countries over years with one DEA model (each country was compared to the best performers among the six years) (hereafter termed 'pooled DEA'). The results from pooled DEA were used to examine the evolution of the performance of national HIV/AIDS programs over time and to conduct the regression analysis, as described below. As these efficiency scores were not adjusted for any factors, we termed them unconditional efficiency scores.2 = 0.20, which permits the use of Tobit model in the study. The Tobit model was built on 120 observations through 2006 because the information on health financing, one of the independent variables, was available only through that year at the time of the analysis (2009). We adjusted the unconditional efficiency scores for those factors, obtaining the conditional efficiency of a country's national AIDS program. We defined a "notable increase" as the change from the lowest quartile to the average value of a variable. Using the Tobit model, we derived the improvement in unconditional efficiency of an AIDS program from "notable" increases in all key determinants. The second stage analysis was conducted with STATA 11.0 After obtaining the efficiency scores from the pooled DEA model, we constructed a random-effects Tobit regression model with finite population adjustment as the second stage of the efficiency analysis ,26,27. TOf all 151 observations, 18% (27 out of 151) were on the production frontier with efficiency scores of 100% using the separate DEA models. The high performers included countries that have been internationally recognized for their successful HIV/AIDS programs such as Thailand , Brazil (2005), and Rwanda (2003). The concordance of our results with empirical observations helps validate our application of DEA to national HIV/AIDS programs and the selection of inputs and outputs. Our highly efficient performers also included some countries that have received less recognition, such as China (2003 and 2006), Chad (2004 and 2006), South Africa  and Paraguay (2006 and 2007). These countries were repeatedly ranked with 100% efficiency. Complete efficiency scores from the DEA models for each country are provided in Additional file The performance of countries' HIV/AIDS programs varied widely. The mean efficiency scores by quartile across 151 observations over the six years were 13.0% in quartile 1 (the worst fourth of observations), 36.4% in quartile 2, 54.4% in quartile 3, and 96.5% in quartile 4 (the best fourth of observations). The overall average output-oriented efficiency was 49.8% (SD = 31.2%), indicating that the countries, on average, could have doubled their outputs if they used money as efficiently as their comparison peers in the same year. When concentrating on inefficient countries alone, the average efficiency score was 38.6% (SD = 23.7%), suggesting a possibility of increases outputs by 2.6 times if they performed well.To illustrate DEA more intuitively, Figure As expected, the results from the pooled DEA model exhibited a wider variation of national HIV/AIDS programs. Eight observations fell on the production frontier with efficiency scores of 100%. They were Thailand 2007), South Africa (2007), Paraguay , China (2006), Peru (2007) and Chad . It was not surprising that the observations on the production frontier were those from recent years because productivity has generally increased over time. Figure 007, SoutTable The positive coefficients for the two interaction terms in the model indicate that fighting corruption would significantly improve the efficiency of using external funds and strengthen the impact of policy implementation on delivering HIV/AIDS services.Our findings also show that a higher share of expenditure from external sources generally improved the efficiency of HIV/AIDS programs. Greater external shares had a higher impact on efficiency in a country with lower levels of corruption. For countries with mid-level efforts in "control of corruption" , a notable increase in the external share of health expenditure (from 1.0% to 5.5%) would result in a 13.5% increase of efficiency.The random-effects Tobit model shows that the relationship between per capita gross national income (GNI) and efficiency followed an inverted U-shape. Efficiency rose with increasing GNI/capita up to the level of I$ 4468. At that level, which corresponds to the income of lower- and middle-income countries in Europe, Central Asia, Latin America and the Caribbean , efficieA 2009 analysis by UNAIDS showed that donors' financial commitments to HIV/AIDS, while impressive, cover only 54% of the resources needed to meet the goal of universal access [We found that the unconditional efficiency of national HIV/AIDS programs was about 50%. This result echoes the previously reported wide variation of efficiency in delivering selected HIV/AIDS services . We noteIf all the countries studied were completely efficient, taking all 151 observations as a whole, their output levels would have increased by 112% for VCT, 65% for PMTCT, and 85% for ARV treatment  and higher health capacities  among countries with higher per capita income, which increases both the demand for and the supply of AIDS services and thus contributes to the high volume of services being provided at lower costs . However, the association between GNI/capita and the efficiency is opposite among countries with GNI/capita > I$ 4468 where, usually, health infrastructure has been in place. Often wages for health personnel and the expected quality of services is higher among wealthier countries ,34. ThesConcerns have been raised as to how best to target the limited resources to poor countries with greater needs for HIV/AIDS . As a suThe main results of this study measure the unconditional efficiency of a country overall in addressing HIV/AIDS, but do not necessarily reflect the performance of the country's health care system specifically, which is often operated under the constraints of a country's general contextual factor (such as the type of epidemics and governance).Critics may question the use of a single production frontier to evaluate all the countries in all years, since countries face different types of epidemics and different political constraints. For example, some countries may perform well because of the favorable environment, such as better governance, while other countries may deliver a mediocre amount of HIV/AIDS services under a harsh environment. When evaluating the efficiency of health care systems in addressing HIV/AIDS, we have to take into consideration countries' environmental factors beyond health sectors by adjusting the efficiency of HIV/AIDS programs for them. The adjustment is analogous to generating multiple production frontiers for the countries to be evaluated. We used the coefficients in Table As evidence for the validating the use of DEA to assess the efficiency of national HIV/AIDS programs, we found that 1) some countries , whose performance is widely respected, received excellent unconditional scores from our analysis; more countries emerge as conditionally efficient after the adjustment for the epidemics and macroeconomic factors, and 2) most coefficients from the Tobit regression bear the expected signs.Our findings are subject to several caveats, however, and should be interpreted with caution. First, the selection of inputs and outputs was limited by data availability. Due to this constraint, we could not include human resources as an input and some key services, such as support to orphans and vulnerable children, as outputs in the model. Furthermore, as all three outputs  are services in health facilities , the uncAs this study concentrated on the technical efficiency of service delivery, we acknowledge that the efficiency of a country's HIV/AIDS program must be interpreted in a broader context of that country, because efficiency is not the only one domain in evaluating a program. Other criteria concern overall effectiveness, as well as economic, political and equity issues.Two future studies would merit special attention. First, a similar study could be expanded to include additional indicators, which might focus specifically on characteristics of the health system rather than the overall society. The current study has shown the feasibility and value of that approach. Second, as efficiency can substantially relieve the financial pressure for scaling up HIV/AIDS services, it would be important to incorporate it into future projections of resource needs for HIV/AIDS.Although efficiency scores of national HIV/AIDS programs are only indicative, given the limitations of the study, our findings suggest that there may be substantial room for improving HIV/AIDS services at the country level with the existing resources. Improving the efficiency of HIV/AIDS services requires actions not only within the health sector, but also in the broader context, such as governance, to ensure that HIV/AIDS funding materializes into services that are targeted to and benefit the population in need. Given the limited resources available for HIV/AIDS services, it is critical to understand countries' performance in delivering HIV/AIDS services to inform strategies to scale up HIV/AIDS services and to help allocate the available funding wisely to combat HIV/AIDS.aWHO, UNAIDS, UNICEF. Towards universal access: scaling up priority HIV/AIDS interventions in the health sector: progress report. 2007.The authors declare that they have no competing interests.All authors have made substantial contributions to the intellectual content of the paper. WZ contributed to the conception and design, acquired the data, analyzed and interpreted the data, and drafted and revised the manuscript. DSS contributed to the conception and design of the study, acquired the data, interpreted the data, and critically revised the manuscript. JC contributed to the critical revision of the manuscript for important intellectual content, and provided technical support. CA contributed to the acquisition of data, interpretation of data, obtaining funding, and providing technical support. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/12/74/prepubcontains additional references [ferences -44.Click here for file


Streptococcus pneumoniae (PNSP), macrolide-resistant S. pneumoniae (MRSP), and macrolide-resistant S. pyogenes (MRGAS) in 20 countries. Total antibiotic use was correlated with PNSP , as was macrolide use with MRSP  and MRGAS . Streptococcal resistance is directly associated with antibiotic selection pressure on a national level.We correlated outpatient antibiotic use with prevalence of penicillin-nonsusceptible  Streptococcuspneumoniae with β-lactam and macrolide use in 12 European countries  and macrolide-resistant S. pneumoniae (MRSP) are markers of resistance to antibiotics commonly used as first-line drugs for respiratory tract infections. Although penicillin-resistant S. pyogenes has never been observed to date, the increasing rates of macrolide-resistant group A streptococci (MRGAS) pose considerable clinical problems in many countries  effect of antibiotic exposure on antimicrobial resistance in streptococci . We also calculated relationships between macrolide use and MRSP for 16 countries , and between macrolide use and MRGAS for 14 countries . For these correlation analyses, we used two-tailed Spearman coefficients (r) for nonparametric correlations.Total use of outpatient antibiotics varied from 9.0 DDD/1,000 inhabitants per day in the Netherlands to 36.5 DDD/1,000 inhabitants/day in France . MacroliS. pneumoniae and S. pyogenes in Europe, North America, and Australia. The ecologic study design chosen allowed us to evaluate the effect of antibiotic consumption on resistance rates on a national level. A strong relationship was documented between total volume of antibiotic consumption and prevalence of PNSP. An almost linear association existed between macrolide use and proportion of MRSP, a biologically plausible finding that has previously been documented on a smaller scale (This report correlates national outpatient data about antibiotic use with prevalence of antibiotic-resistant S. pneumoniae and other streptococci (We found large differences in resistance rates, even in neighboring countries. A variety of factors are responsible for this, but the selective pressure exerted by inappropriately used antibiotics is likely the most important (Antibiotic use exerts selective pressure on resistance in respiratory pathogens in several ways (This ecologic study has some limitations. First, antibiotic sales data cannot be used synonymously with antibiotic exposure without highlighting the problem of patient compliance, which is very difficult to quantify. However, in case of low compliance, antibiotics may be used later, and this self-medication may also contribute to resistance. Second, large variations in resistance are typically found, depending on the examined age group, with highest resistance rates and largest amounts of antibiotic consumption usually in children. Due to the study design chosen, we could not account for these differences. Third, no single surveillance study was identified that provided sufficient data on national resistance rates for all countries. Therefore, considerable heterogeneity was found among the quality and characteristics of the included surveillance studies. We tried to minimize this bias by excluding publications not meeting certain standards or examining only selected patient populations. Fourth, the type and number of tested organisms varied widely. Thus, sampling bias may have influenced the results of this study. Finally, data are lacking regarding the maximum time lag between antibiotic consumption in the community and possible changes in resistance patterns on a national level. A period of 1 to 2 years seems plausible, although more rapid changes can be observed.Further studies should be undertaken to extend our analyses to Asian countries that are demonstrating high rates of streptococcal resistance (


Inappropriate prescription of antibiotics may contribute towards higher levels antimicrobial resistance. A key intervention for improving appropriate antibiotic prescription is surveillance of prescription. This paper presents the results of a longitudinal surveillance of broad-spectrum antibiotic prescription in 5 public-sector hospitals in Singapore from 2006 to 2010.Quarterly antibiotic prescription data were obtained and converted to defined daily doses (DDDs) per 1,000 inpatient-days. The presence of significant trends in antibiotic prescription over time for both individual and combined hospitals was tested by regression analysis and corrected for autocorrelation between time-points. Excluding fluoroquinolones, there was a significant increase in prescription of all monitored antibiotics from an average of 233.12 defined daily doses (DDD)/1,000 inpatient-days in 2006 to 254.38 DDD/1,000 inpatient-days in 2010 . Increasing utilization of carbapenems, piperacillin/tazobactam, and Gram-positive agents were seen in the majority of the hospitals, while cephalosporins were less prescribed over time. The combined expenditure for 5 hospitals increased from USD9.9 million in 2006 to USD16.7 million in 2010.The rate of prescription of broad-spectrum antibiotics in Singaporean hospitals is much higher compared to those of European hospitals. This may be due to high rates of antimicrobial resistance. The increase in expenditure on monitored antibiotics over the past 5 years outstripped the actual increase in DDD/1,000 inpatient-days of antibiotics prescribed. Longitudinal surveillance of antibiotic prescription on a hospital and countrywide level is important for detecting trends for formulating interventions or policies. Further research is needed to understand the causes for the various prescription trends and to act on these where necessary. Antibiotics are an invaluable shared resource that is at risk of becoming both scarce and less effective in the face of a diminishing antibiotic pipeline and rising antimicrobial resistance High antimicrobial resistance rates are prevalent in Singaporean hospitals p = 0.025), although the correlation coefficient (R2) was low at 0.28. Inclusion of the fluoroquinolones – which as a class comprised more than 60% of all antibiotics monitored – rendered the results non-significant. The increase in prescription of carbapenems, piperacillin/tazobactam, polymyxins and Gram-positive agents was balanced by a decrease in cephalosporin prescription and non-significant trend towards reduction of prescription of fluoroquinolones.Broad-spectrum antibiotic prescription results according to antibiotic class are depicted in The trends in prescription of individual antibiotics within major antibiotic classes are depicted in Trends in actual expenditure on antibiotics monitored per 100 inpatient-days are shown in This work adds to the available sparse literature on country-level reports of hospital antibiotic consumption Staphylococcus aureus , extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae  and carbapenem-resistant Pseudomonas aeruginosa  have not increased over the five-year period. It is plausible that with greater awareness of these pathogens, clinicians were progressively prescribing more broad-spectrum antibiotics despite the lack of change in resistance trends. The issue of inappropriate prescription had been examined recently – the results with regards to carbapenem usage are consistent with findings in hospitals elsewhere However, local rates of methicillin-resistant th-generation cephalosporin prescription decreased even as piperacillin/tazobactam prescription increased in three hospitals. In Hospital 3, this had been actively encouraged in an effort to decrease the rates of ESBL-producing Enterobacteriaceae by decreasing cephalosporin selection pressure. The cause(s) for the switch in the other hospitals is less clear, although more aggressive marketing by the pharmaceutical industry and/or the direct impact of antimicrobial stewardship programs (ASPs) that had been established in Hospitals 1-3 between 2008 and 2009 cannot be ruled out as contributing causes. Ertapenem and meropenem prescription had increased at the cost of imipenem prescription – dosing advantages in the former and the perceived advantage of reduced neurological adverse effects in the latter had impelled the shift in prescription trends. In Hospitals 1 and 3, ertapenem had been promoted over the other carbapenems because of its lack of selective pressure on carbapenem-resistant non-fermenting Gram-negative bacteria Several antibiotics are natural substitutes of each other. Third- and 4The increase in expenditure on monitored antibiotics over the past 5 years reflects the actual increase in DDD/1,000 inpatient-days of antibiotics prescribed. The primary reason is that there was an increase in prescription of more expensive antibiotics  whereas prescription of some of the cheaper generic antibiotics  had decreased. Among the three hospitals with ASPs, broad-spectrum antibiotic expenditure failed to increase only in Hospital 1, but this was the only institution where the ASP had covered all inpatient wards by 2010 There are a number of limitations of this work. Firstly, we did not correlate antibiotic usage with resistance. However, this had been attempted previously with few significant results In conclusion, longitudinal surveillance of antibiotic prescription on a hospital and countrywide level is important for detecting trends for the ultimate basis of formulating interventions or policies. Further research is needed to understand the causes for the various prescription trends and to act on these where necessary. It is important to note that both surveillance without intervention and intervention without surveillance are inefficient practices.This was a longitudinal multicenter database surveillance study carried out over a period of 5 years from 2006 to 2010. Five of 6 public sector hospitals in Singapore participated in the study – the sixth was a maternal and child hospital that was excluded because the large pediatric population would affect measurements of antibiotic prescription. There were 2 tertiary hospitals  and 3 secondary hospitals  ranging between 400 to 1,600 beds in size.rd and 4th generation cephalosporins , fluoroquinolones , β-lactam/β-lactamase inhibitors (piperacillin/tazobactam), Gram-positive agents  and others . Denominator data in the form of hospital inpatient-days, i.e. the sum of each daily inpatient census every quarter, were obtained from each hospital's administrative records. The defined daily dose (DDD) per 1,000 inpatient-days for each drug and drug category prescribed every quarter was calculated following the World Health Organization (WHO) anatomical therapeutic chemical (ATC) classification of 2011 Antibiotic prescription data were extracted from each hospital's electronic pharmacy records. These figures comprise actual prescription data rather than purchase data. The antibiotics tracked included the carbapenems , 3p-value of ≤ 0.05 and coefficient of determination (R2) greater than 0.3. Because tigecycline, daptomycin and doripenem were launched in Singapore within this period  and prescription volumes were low, these antibiotics were not analyzed individually.All statistical analyses were performed using Stata 11.0 . The presence of significant trends in antibiotic prescription over time for both individual and combined hospitals was tested by regression analysis and corrected for autocorrelation between time-points by using the Cochrane-Orcutt estimation following determination of the Durbin-Watson statistic. A trend was considered significant if the regression analysis showed a


SSALC was established to characterize HIV/AIDS-related lymphoma and the indigenous background of malignant lymphomas (ML) in sub-Saharan Africa. Because WHO classified lymphoma subgroups can vary in prevalence African, Asian or European ancestry, we surveyed lymphoma heterogeneity in geographically diverse East, South and West sub-Saharan populations, particularly for HIV/AIDS associated immunophenotypes.kappa and lambda light chains (CISH) and c-myc and bcl2 translocations (FISH). HIV/AIDS diversity controls were contributed from Europe by consortium and USA by ACSR.A consortium of African pathologists, hematologist/oncologists and oncologic surgeons contributed ML cases and participated in sub-grouping according to WHO classification criteria after appropriate Institutional Review Board (IRB) approvals, Memoranda of Understanding and Material Transfer Agreements were obtained. Paraffin blocks were examined for tissue morphology (H&E), immunophenotype (34 antibodies IHC), EBER, Consortium members contributed 46 - 368 cases each with 1408 total cases to date: 246 diffuse large B-cell lymphoma (DLBCL), 296 Burkitt lymphoma, 163 Hodgkin disease, 69 plasma cell proliferative disorders and 644 others. Aggressive DLBCL, plasmacytoma/plasmablastic lymphoma, KSHV disease and lymphoid hyperplasia will be highlighted.Sub-Saharan Africa has a variety of ML subgroups; true incidence altered by: 1) Aspiration vs. biopsy for diagnosis; 2) HIV status not communicated to pathologist; 3) known HIV/AIDS patients not biopsied; 4) initial diagnosis by morphology alone, 5) tissue preservation/processing variable.. General observations: HIV/AIDS-related lymphoma is more likely EBER+, has higher cell proliferation rates, and unfavorable immunophenotypes; regions differ in HIV clades with South (clade C) having the most “immunosuppression” associated lymphoma subgroups; East region has more pre-T lymphoblastic lymphomas and West region has more follicular lymphomas. Confounders: infectious lymphadenopathies (EBV+ lymphoproliferations), undifferentiated neuroblastomas, neuroectodermal tumors (PNETs), poorly differentiated, metastatic carcinomas and malignant melanoma (amelanotic).