Several organizations and individual authors have been proposing quality indicators for the assessment of clinical care in HIV/AIDS patients. Nevertheless, the definition of a consensual core set of indicators remains controversial and its practical use is largely limited. This study aims not only to identify and characterize these indicators through a systematic literature review but also to propose a parsimonious model based on those most used.MEDLINE, SCOPUS, Cochrane databases and ISI Web of Knowledge, as well as official websites of organizations dealing with HIV/AIDS care, were searched for articles and information proposing HIV/AIDS clinical care quality indicators. The ones that are on patient’s perspective and based on services set were excluded. Data extraction, using a predefined data sheet based on Cochrane recommendations, was done by one of the authors while a second author rechecked the extracted data for any inconsistency.A total of 360 articles were identified in our search query but only 12 of them met the inclusion criteria. We also identified one relevant site. Overall, we identified 65 quality indicators for HIV/AIDS clinical care distributed as following: outcome (n=15) and process-related (n=50) indicators; generic (n=36) and HIV/AIDS disease-specific (n=29) indicators; baseline examinations (n=19), screening (n=9), immunization (n=4), prophylaxis (n=5), HIV monitoring (n=16), and therapy (=12) indicators.There are several studies that set up HIV clinical care indicators, with only a part of them useful to assess the HIV clinical care. More importantly, HIV/AIDS clinical care indicators need to be valid, reliable and most of all feasible. Quality performance or quality of clinical care is the main subject in the health care system and it can be precisely defined. Many studies have been made on this subject -3. The IMainz says that for developing evidence-based clinical indicators, it is necessary to follow certain steps, namely choosing the clinical area to evaluate, organizing the measurement team, providing an overview of existing evidence and practice, selecting clinical indicators and standards, designing measure specification, and performing a pilot test .The pilot test aims to identify areas that require further specifications of the quality measures. The validation process determines the degree to which an indicator measure, what is intended to measure, that is, whether the results of measurement corresponds to the true state of the phenomenon being measured , Figure For each quality indicator it is important to verify if it meets quality requirements such as validity, reliability, relevance and applicability, based evidence ,10, and The assessment of the quality of care given to HIV/AIDS patients has been a major focus of the HIV/AIDS disease issue since 1990. Agins et al.  proposedIn this study we performed a systematic review to identify the current existing quality indicators, evidence-based, used for monitoring and evaluating HIV/AIDS impatient and outpatient clinical care, and, based on the clinical relevance and practice utility, we propose a core set of indicators, through observational studies.A systematic review (SR) of observational studies on HIV/AIDS quality indicators for clinical care and its development and validation methods, using electronic databases such as MEDLINE (1966–2012 through PubMed), SCOPUS (2004–2012), Cochrane Collaboration database (CENTRAL) (1980–2012) and ISI Web of knowledge ( −2012), without limitations of language. Additional studies were identified by searching reference-lists of articles and on the official institutions sites that work with HIV/AIDS quality indicators.We used the following search terms and Medical Subject Headings (MeSH) to search observational studies: “observational studies”, “quality indicators”, “validation studies”, “HIV/AIDS”. We developed a search query for each database, and one of the 3 search-queries we set up down can be seen in Table In this SR we included the primary studies that had met the following criteria, (i) talk about HIV/AIDS clinical care quality indicators, and (ii) set up a conceptual model of quality indicators for HIV/AIDS in clinical care context, or (iii) describe or validate HIV/AIDS clinical care quality indicators.We excluded studies that (i) are based only on services set, (ii) are based on patient perspective or (iii) did not provide sufficient details in methods and results sections, thus failing to answer research questions.Eligibility assessment was performed independently by 2 reviewers based on inclusion criteria, and disagreements between reviewers were solved by consensus.We developed a data extraction sheet adapted to observational studies (based on Cochrane Consumers and Communication review Group’s data extraction template), we pilot-tested it on 2 included studies, and refined it accordingly. One reviewer extracted the following data from included studies and the second checked the extracted data: first author, year, publication country and study design, objective, number of clinical indicators and selection criteria. For each indicator: name, type (process or outcome), quality (generic or specific) selection and validation criteria. Disagreements were solved by discussion between the two reviewers. If no agreement could be reached, a third author was planned but it was not necessary. We did not contact any authors for further information.For each quality indicator of HIV clinical care some parameters were determined. Similar indicators were grouped together. We classified indicators according to the dimension of quality of care as process , or outcome  indicators as described by Donabedian . We alsoThe search of MEDLINE, Cochrane, Scopus and ISI web knowledge provided a total of 360 citations. After adjusting for duplicates 343 remained. Of these, 329 studies were discarded after revision as the papers clearly did not met the inclusion criteria. One study, Bennet 1996) [96 [19] wOn the internet, we looked for official sites and only one met the inclusion criteria, namely the Indicator Registry  owned byThis systematic review covers a range of 23 years between the first and the last study, published in 2012. Nine studies represent experiences made in the USA , one in From the 12 studies, 65 HIV clinical care quality indicators related to process and outcome were retrieved. From these 65 indicators, 29 are HIV/AIDS-specific and 36 are generic indicators; 15 are related to outcome and 50 to process indicators. These indicators cover the following clinical care areas: Baseline evaluation (19), screening for opportunistic diseases and STD (9), immunization (4), prophylaxis (5), HIV monitoring (16), and therapy (12). The boundaries of these clinical areas are not very well delimitated, and therefore some indicators could be easily changed from one to another area . One of the challenges hospitals face nowadays is the quality of the data they collect -38. SomeWhen we analyze the validity of the quality indicators, a common characteristic is that most of the studies assess the quality of care, only one is about the validity of the quality indicator itself. So for each indicator, it is necessary to analyze it in order to find out its reliability, relevance and applicability.Indicators included in the WHO Indicator and Measurement Registry are set up “on a national basis”; some of them can be applied at a local level, but few of them are to assess the quality of HIV clinical care. These indicators are well defined and are ready to be used by everyone as they are valid and reliable.Many institutions have been using HIV/AIDS quality indicators to assess the quality of clinical care and make a benchmarking between caregivers. The results seem to be good, and improvement of care has been achieved ,26.Quality care indicators or clinical care indicators are used in many situations, as the ones described next. (1) To improve the quality of care, for instance Asch at al. , used a The next step is to test the measures in real data, to find out the reliability of each indicator and to validate a set core of quality indicators that can be used to assess HIV clinical care and may be used to compare services, hospitals or even countries.This review had several limitations. Although we found few studies, we made a wide search on the web using multiple terms and databases. Search in gray literature was limited. Studies in languages other than English were not included. Anyway, as far as we know, there is no other published systematic review about HIV/AIDS quality indicators for clinical care assessment, and we summarized here a vast and wide set of information. The fact of the matter is that we include experiences from USA, Ethiopia, Malawi, Spain and Thailand, and a core of organizations that work on the HIV/AIDS field under the WHO umbrella.We wanted to identify a core set of indicators that represent optimal care and that will facilitate uniform measurement and benchmarking of the quality of HIV/AIDS clinical care at a local level, and can lead to the establishment of comparative reporting between developed and developing countries.The main findings of this systematic review are that there are efforts from the USA and Spain to establish a national core set of indicators useful to assess the HIV/AIDS clinical care; the assessment of HIV/AIDS clinical care must take into account the main aspects of baseline assessment, screening, immunization, prophylaxis, HIV monitoring, and therapy. Most of the indicators are process-related, are intended to guide (the good practice) and to evaluate the clinical care provided to HIV/AIDS patients.The authors declare that no competing interests exist.EC carried out the systematic review, conceived the study, and participated in its design and coordination and drafted the manuscript. VC participated in data collection process. AF participated in the analysis of the results and helped to draft the manuscript. CC participated in the design and coordination of the study. AS participated in the design and coordination of the study. ACP conceived the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/13/236/prepub


The preferred choice of the best surgical antibiotic prophylaxis practice is usually low despite many published guidelines. This study describes the application of quality indicators for antibiotic prophylaxis in a hospital in Sao Paulo, Brazil.A retrospective study was carried out from November 2009 to March 2010. Medical records from adult inpatients submitted to cardiac, neurological and orthopedic clean surgeries were included. The quality indicators for antibiotic prophylaxis conformity were validated in a previous study. They were composed of six parameters of adequacy as stated by the Hospital Infection Control Committee (HICC) guideline. The conformity index was considered 100% when the antibiotic prophylaxis showed adequacy in all parameters evaluated. We investigated the association between conformity in each parameter and selected population characteristics. Analyses were conducted with 5% significance.Medical records from 101 (13.5%) cardiac, 128 (17.1%) neurosurgical, and 519 (69.4%) orthopedic surgeries were evaluated. The general conformity index was 4.9%. The greatest number of surgeries where antibiotic prophylaxis was used without HICC indication occurred among orthopedic procedures (n=293). The total conformity index was 5.8%, 3.1% and 3.0% respectively for orthopedic, neurological and cardiac surgeries. The parameter of administration via was the best achieved; the parameter of duration was the worst. No association was identified between parameter conformity and population characteristics.This study reveals a low level of acceptance for HICC guidelines for antibiotic prophylaxis. Quality indicators should be fed back to surgical teams on a regular basis to improve surgical prophylaxis.None declared.


In order to better understand the molecular mechanisms of 1BnTIQ—produced toxicity, we examined the impact of different concentrations of 1BnTIQ  on glutamate-induced apoptotic pathway. We measured the markers of apoptosis, such as caspase-3 activity, lactate dehydrogenase release, and mitochondrial membrane potential. Molecular data were supported at the cellular level by calcein AM and Hoechst 33342 staining. The obtained data demonstrated concentration-dependent effects of 1BnTIQ opposing apoptosis, and evidenced that 1BnTIQ in a low concentration (50 μM) exhibited neuroprotective activity, whereas in 10 times higher concentration (500 μM) might be neurotoxic, and significantly intensified glutamate-induced increase in apoptosis markers. Additionally, using an ex vivo molecular study we indicated that both acute and chronic administration of 1BnTIQ did not affect the level of alpha synuclein and tyrosine hydroxylase protein in the rat substantia nigra. Summarizing the studies, we suggest that 1BnTIQ is a rather weak endogenous neurotoxin; however, it should be taken into account that in higher μmoles concentrations, it can initiate apoptosis in the central nervous system and may be involved in the etiopathology of neurodegenerative diseases. The programed cell death (apoptosis) is a biological process in which unwanted and/or damaged cells are killed, so that the organism could maintain homeostasis. The apoptosis process is a characteristic not only of normal development but also of numerous neurodegenerative diseases and neurological conditions, such as Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease, and stroke  is an endogenous neurotoxin which has been proposed to be one of the etiological factors of idiopathic PD  (22 °C) under an artificial light/dark cycle .) once or chronically for 14 consecutive days. Control rats were treated with an appropriate solvent. The rats were killed by decapitation 3 h (caspase-3 activity) or 24 h (α-synuclein and tyrosine hydroxylase) after the last drug injection, and different structures of the brain were dissected. The experiments were carried out between 9.00 and 16.00 h.The rats were administered 1BnTIQ at a dose of 50 mg/kg intraperitoneally  at the Department of Drug Chemistry of the Institute of Pharmacology, the Polish Academy of Sciences in Kraków. Purity of the compound was verified by measurement of its melting point; and homogeneity was assessed on a chromatographic column. The compounds were dissolved in a 0.9 % NaCl solution. Glutamic acid was purchased from Sigma-Aldrich , whereas calcein AM and Hoechst 33342 were purchased from Molecular Probes .5 cells per cm2 onto poly-ornithine (0.01 mg/ml)-coated multiwell plates. The cultures were maintained at 37 °C in a humidified atmosphere containing 5 % CO2 for 7 days in vitro (DIV) prior to experimentation. The level of astrocytes, as determined by the content of intermediate filament protein GFAP , did not exceed 10 % for all cultures  at 15th–17th day of gestation and were cultured essentially as described earlier , 1BnTIQ , or glutamate and 1BTIQ for 24 h. To avoid unspecific effects in our study, 1BnTIQ was used in the concentrations which did not affect the control level of caspase-3 activity and lactate dehydrogenase (LDH) release. All the compounds were originally dissolved in dimethyl sulfoxide (DMSO), and then further diluted in culture medium to achieve DMSO concentrations below 0.1 %.Apoptotic cells were detected by Hoechst 33342-staining 24 h after initial treatment, as described previously . The amounts of p-nitroanilide were monitored continuously over 60 min with a Multiskan Spectrum Microplate Spectrophotometer . Data were analyzed with Ascent software, normalized to the absorbance in vehicle-treated cells, and expressed as percent of control ± SEM of three to four independent experiments. The absorbance of blanks, acting as no enzyme controls, was subtracted from each value.Caspase-3 activity was determined according to Nicholson et al. , using sTo quantify cell death, LDH released from damaged cells into the cell culture media was measured 24 h after treatment with glutamate and 1BnTIQ . LDH release was measured as previously described  originating from 1-BnTIQ or solvent-treated rats were weighted, homogenized on ice in 20 volumes of RIPA buffer  with addition of protease inhibitors mixture (Pierce). Protein concentration in the supernatants was determined using bicinchoninic acid protein assay kit (Pierce). Afterward, the samples containing 5 μg of total protein were fractionated by 12 or 10 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), as described previously by Laemmli  and procThe amounts of protein per lane as well as antibody concentrations were optimized in pilot studies so that at least threefold differences in protein content were linearly reflected on immunoblots.The signals were visualized and quantified by the densitometric analysis with the FUJI-LAS 4000 system and Fuji Multi Gauge software. Results are presented as a percentage of the control of the analyzed protein: β-actin ratio ± SEM.p < 0.05; **p < 0.01; ***p < 0.001 (vs control group); #p < 0.05; ##p < 0.01; ###p < 0.001 (vs glutamate group).Statistical tests were performed on raw data expressed as the mean arbitrary absorbance or fluorescence units per well containing 50,000 cells . A one-way analysis of variance (ANOVA) was used to determine overall significance. Differences between control and experimental groups were assessed with the post hoc Neuman–Keuls test, with the significant differences marked in the following way: *Statistical significance of the data from Western blot analysis was assessed using a one-way ANOVA followed (if significant) by the Tukey test for post hoc comparison with the control  group.p < 0.001)  intensified the effect of glutamate and produced elevation of caspase-3 activity by 100 %   intensified glutamate-induced release of LDH by 190 %  Fig. . 1BnTIQ 0 % Fig. .Fig. 2ThIn the cells treated with 50 and 100 μM 1BnTIQ, the activity of caspase-3 was ~110 % Fig. . Exposurp < 0.001)  caused an about 70 % loss of mitochondrial membrane potential in the neuronal cells (01) Fig. . Co-trea01) Fig. . The adm01) Fig. .Fig. 3ThA continuous 24-h exposure of hippocampal cultures to glutamate (1 mM) reduced the density of living cells, as indicated by the decreased number of cells with light-colored cytoplasm  increase in this parameter (by ~90 %)  administration of 1BnTIQ induced a significant  did not change the level of tyrosine hydroxylase in the substantia nigra measured 24 h after the last does Fig. .Fig. 7ThThe main finding of this paper is that 1BnTIQ produced concentration-dependent opposite effects on markers of apoptosis evaluated by in vitro studies in primary hippocampal cultures of the mouse and the rat. 1BnTIQ in a low concentration (50 μM) possessed neuroprotective activity, while in a 10 times higher concentration (500 μM) might be neurotoxic, and significantly intensified glutamate-induced neurotoxicity. On the other hand, in vivo chronic administration of 1BnTIQ did not produce neurotoxic effects on dopamine neurons in the substantia nigra as assessed by the levels of two proteins: tyrosine hydroxylase and alpha-synuclein.1BnTIQ is an endogenous neurotoxin which has been proposed to be an etiological factor of PD  of glutamate destroyed neurons  but not acute administration of 1BnTIQ (50 mg/kg i.p.) produced a significant elevation of caspase-3 activity in the rat hippocampus Fig.  or tyrosThe present data confirm our earlier suggestion that 1BnTIQ is an endogenous neurotoxin with a rather weak toxicity (Wąsik et al. In conclusion, our study strongly indicates that 1BnTIQ in primary hippocampal cultures may exert opposite effects on markers of apoptosis depending upon its concentration: neuroprotection at low concentrations or pro-apoptotic effect at higher concentrations. However, 1BnTIQ in a high concentration and upon prolonged exposure leads to apoptotic cell death and can be one of the etiological factors of neurodegenerative disease.


Infant dietary exposures have been linked to type 1 diabetes (T1D) development. IgG4 antibody responses to food antigens are associated with food intolerances but have not been explored prospectively in the period preceding T1D.Using a case-cohort design, IgG4 antibodies to ß-lactoglobulin, gluten, and ovalbumin were measured in plasma collected annually from 260 DAISY participants. Of those, 77 developed islet autoimmunity (IA), defined as positive for either insulin, GAD65 or IA-2 autoantibodies on two consecutive visits, and 22 developed T1D.In mixed model analysis adjusting for HLA-DR status, T1D family history, age and ethnicity, higher ß-lactoglobulin IgG4 concentrations were associated with shorter breastfeeding duration  and earlier first cow’s milk exposure . Higher gluten IgG4 was associated with older age at gluten introduction . In proportional hazards analysis adjusting for HLA-DR status, T1D family history and ethnicity, IgG4 against individual or multiple dietary antigens throughout childhood were not associated with IA. In addition, mean antigen-specific IgG4 concentrations in infancy (age <2 years) were not associated with risk of IA nor progression to T1D. Higher ovalbumin IgG4 at first IA positive visit was marginally associated with progression to T1D .We found no association between the IgG4 response to β-lactoglobulin, gluten, and the development of either IA or T1D. The association between higher ovalbumin and progression to T1D in children with IA should be explored in other populations. Type 1 diabetes (T1D) is an autoimmune disease preceded by a period of sub-clinical islet autoimmunity (IA) The infant diet can affect the permeability of the immature gut. Breastfeeding has been shown to tighten epithelial junctions, and thus decrease intestinal permeability in infants Circulating IgG antibody concentrations may be an indirect measure of intestinal permeability. In case-control studies, T1D has been associated with increased IgG antibodies to ß-lactoglobulin, a cow’s milk protein In a prospective cohort of children with increased genetic risk for T1D, we looked for an association between a generalized immune reaction to common infant diet exposures and IA and T1D development. We measured concentrations of IgG4 antibodies, a subclass of IgG associated with the gastrointestinal mucosa The Colorado Multiple Institutional Review Board approved all study protocols. Informed written consent was obtained from the parents/legal guardians of all children. Assent was obtained from children age ≥7 years.The Diabetes Autoimmunity Study in the Young (DAISY) is a prospective study of two groups of young children at increased risk for developing T1D. The DAISY study has enrolled approximately 2,500 children at increased risk for type 1 diabetes from 1993 to 2006. The details of the newborn screening Prospective follow-up of DAISY children included clinic visits at 9, 15 and 24 months (if child enrolled at birth) or at enrolment visit (if child enrolled later in childhood), and annually thereafter up to age 15 years . The sub-cohort was selected from the DAISY cohort via stratified random sampling based on HLA-DR genotype and T1D family history. This representative sub-cohort was used to test the internal consistency of the IgG4 antibodies by correlating them with infant diet exposures.Based on availability of biological samples and timing of entry into the study, 1,433 DAISY children were eligible for selection into a representative sub-cohort of 192 children .To explore the IA outcome, we conducted a case-cohort analysis. The representative sub-cohort, which contained 9 children who developed IA, was supplemented with 68 DAISY children from outside the sub-cohort who developed IA. Thus, the IA analysis cohort includes 77 children that developed IA, and 183 children that did not develop IA .To explore the T1D outcome, we examined whether the IgG4 concentrations at the first IA positive visit were associated with progression to T1D in IA positive children. Of the 77 IA positive children in the case-cohort analysis, 22 subsequently developed T1D, as of December 2011 IgG4 antibodies to ß-lactoglobulin, gluten, and ovalbumin were measured in plasma collected throughout childhood. Blood drawn from DAISY subjects is kept from light at all times. Plasma is separated immediately, snap frozen in liquid nitrogen, and stored at −70 degrees Celsius until being sent for analysis. All available plasma samples on the 192 children in the representative sub-cohort were tested for IgG4 antibodies. The 68 children that developed IA outside of the representative sub-cohort also were tested for IgG4 antibodies on multiple visits throughout infancy and childhood, but following case-cohort methodology, only the IgG4 antibody results at the first IA positive visit  were used in the time-varying survival analysis.Plasma samples were sent to National Jewish Health in Denver, Colorado for IgG4 assay. β-lactoglobulin, gluten, and ovalbumin ImmunoCAPs  tests were run on a Phadia ImmunoCAP 250 instrument. For analysis, values below the lower limit of detection for specific IgG4 were replaced by half the lower limit of detection (0.01 mgA/L). The intraclass correlation coefficients (ICC) from 18 blinded duplicate samples were 0.992 for β-lactoglobulin, 0.993 for gluten, and 0.994 for ovalbumin. The coefficients of variation were 1.9% for β-lactoglobulin, 2.6% for gluten, and 2.2% for ovalbumin.The following demographic and cohort selection variables were collected at enrolment interview: gender, race/ethnicity, maternal education, gestational age, and T1D status of first degree relatives. Breastfeeding duration and child’s age at introduction of foods and milks/formulas containing cow’s milk, gluten (wheat and barley – no child was exposed to rye in infancy), and eggs were collected via parent interviews every three months until 15 months of age.Due to three extreme outliers, the β-lactoglobulin IgG4 concentrations were winsorized  prior to analysis. There were no extreme outliers of gluten or ovalbumin IgG4 concentrations. There are no accepted cut-off values for an ‘increased’ IgG4 response to any of the three antigens that we are analyzing. Therefore, we analyzed the IgG4 concentrations as continuous variables, as quartiles, and as means. In order to test the hypothesis that a generalized antigenic response may be associated with IA and progression to T1D, we designed a composite measure of antigenic response (‘quartile rank of dietary antibodies’), in which concentrations of each IgG4 were divided into quartiles, ranked 1 (‘low’) through 4 (‘high’), and summed for each clinic visit  We also examined whether mean IgG4 concentrations prior to age 2 years were associated with risk of developing IA and T1D. Finally, because children who have developed transglutaminase antibodies may have a damaged epithelial lining, each analysis included a sub-analysis in which we excluded the dietary antibody measurements from children that developed transglutaminase antibodies at any time during follow-up. to examine the internal consistency of our dietary antibodies by testing whether relevant infant dietary exposures were associated with IgG4 antibody concentrations to ß-lactoglobulin, gluten, and ovalbumin throughout childhood. Dietary exposures tested (independent variables) were exclusive and total breastfeeding duration, and age at introduction to formulas, milks and foods containing cow’s milk, gluten, and eggs, in separate models. Due to non-normal distributions, we log transformed the IgG4 dietary antibodies before analyzing them as dependent variables. A mixed model was used to analyze infant dietary exposures as predictors of dietary IgG4 concentrations. Models were adjusted for HLA, T1D family history, ethnicity, age, and IA status.We conducted three sets of analyses. First, we used the representative sub-cohort  to examine the association between IgG4 dietary antibody concentrations (independent variables) and time to IA development (dependent variable). Other variables of interest that were tested included exclusive and total breastfeeding duration and age at introduction to formulas/milks and foods containing cow’s milk, gluten, and eggs. Risk estimates were calculated by weighted Cox proportional hazards regression models, which accounts for right censoring, using the Barlow method The second set of analyses used the case-cohort The third set of analyses was conducted on all IA cases , a first degree relative with T1D (52% vs. 36%), and/or a mother with >12 years of education (86% vs. 70%). No significant differences were found between children that did and did not develop IA in terms of sex, ethnicity, maternal age at birth, exclusive or total breastfeeding, or age at introduction to cow’s milk, gluten, or eggs.In the representative sub-cohort, concentrations of IgG4 antibodies to ß-lactoglobulin, gluten, and ovalbumin were correlated with each other .In the representative sub-cohort, IgG4 concentrations of ß-lactoglobulin were significantly inversely associated with total breastfeeding duration : −0.05, −0.006) and marginally inversely associated with age at first cow’s milk exposure . Increased IgG4 gluten antibody concentrations were marginally associated with later age at gluten introduction . Sex, ethnicity, gestational age, and maternal age at child’s birth were not associated with concentrations of any of the three dietary antibodies.In survival models adjusted for HLA, T1D family history, and ethnicity, IgG4 concentrations of β-lactoglobulin, gluten and ovalbumin throughout childhood were not associated with risk of IA Table 1.In survival models adjusted for HLA, family history of T1D, ethnicity, and age at first positive IA visit, higher ovalbumin IgG4 antibody concentrations at IA onset were marginally associated with more rapid progression to T1D (p = 0.05). In separate models, concentrations of IgG4 antibodies to ß-lactoglobulin and gluten were not associated with progression to T1D Table 2.In order to eliminate the influence of the deleterious process marked by the presence of transglutaminase antibodies, we re-analyzed the IgG4 antibody data with just the records for the children that were always negative for transglutaminase antibodies (cut-off for positivity >0.050) .The results described above did not change substantially after elimination of the transglutaminase positive children, except to make the association between older age at introduction to gluten and higher gluten IgG4 antibody concentrations in childhood non-significant. Also, after removing the thirteen TG+ subjects (three of whom developed T1D) from the T1D analysis, the association between ovalbumin antibody concentrations and T1D development became marginally significant (p = 0.06) In healthy children, overall IgG4 antibody concentrations drop from birth to a nadir around 6 months of age as maternal IgG4 concentrations in the infant’s system decline. IgG4 concentrations usually increase as infants and young children are exposed to allergen-containing foods, then plateau around age 6–8 years We hypothesized that increased IgG4 concentrations against multiple dietary antigens, indicating increased gut permeability and subsequent mucosal immune response, would be associated with earlier IA development, and more rapid progression to T1D in children with IA. We approached this research question in three ways. First, we analyzed the IgG4 concentrations throughout childhood (as time-varying covariates). We then explored a combined measure of antigen response by ranking and summing quartiles of IgG4 concentrations of the three IgG4 antibodies. Finally, we examined infancy as a critical time period.Before analyzing IA and T1D risk, we defined the association between relevant infant dietary exposures and childhood IgG4 concentrations to ß-lactoglobulin, gluten, and ovalbumin. In our cohort of children with increased genetic risk for T1D, circulating concentrations of the three dietary IgG4 antibodies measured  were strongly correlated with each other. We found that ß-lactoglobulin concentrations were inversely associated with total breastfeeding duration and with age at introduction to cow’s milk. This finding is consistent with other studies, in which early exposure to cow’s milk in infancy was associated with higher IgG antibody concentrations throughout infancy and early childhood et al. et al. examined ß-lactoglobulin levels in the first 3 years of life. Also, Luopajarvi and colleagues measured ß-lactoglobulin IgG, while we measured the IgG4 isotype.Previous research has found T1D to be associated with increased IgG antibodies to ß-lactoglobulin et al. found an immune response to dietary wheat polypeptides and gliadin in transglutaminase-negative young adults with long duration T1D Gluten consumption has been shown to initiate an inflammatory response in the gut, which may compromise intestinal barrier function and increase gut permeability Our study found higher ovalbumin IgG4 antibody concentrations at the first IA positive visit was marginally associated with faster progression to T1D. Our prospective study results confirm the findings of two previous case-control studies, which found higher IgG antibodies to ovalbumin in T1D patients The immune factors influencing autoimmune disease risk are complex. In our study, the increased ovalbumin IgG4 concentrations may indicate an immunological tolerance to foreign proteins, resulting from a greater Th2 response Our finding of a significant association between T1D risk and ovalbumin IgG4 concentrations, but not β-lactoglobulin or gluten IgG4 concentrations, does not agree with our hypothesis of a greater generalized mucosal immune response, indicating increased intestinal permeability, in children that develop T1D. Our discrepant finding may be due to the greater potency of the ovalbumin antigen. IgG4 antibodies to ovalbumin can be found in infants prior to egg exposure Our mostly negative findings may also be due to the very gradual rise in IgG4 antibody concentrations throughout childhood: IgG4 concentrations do not reach adult levels until age 13 years Strengths of this study include the prospective, longitudinal nature of the data, with frequent follow-up intervals and collection of exposure variables prior to IA or T1D onset. No other studies have prospectively examined the association between IgG4 antibodies to multiple dietary factors and earlier IA or T1D development. The case-cohort analysis structure allowed us to efficiently conduct a resource-intensive analysis on a sub-sample of the full cohort, without compromising the strength of the prospective cohort design. Limitations of this analysis include lack of generalizability to the US pediatric population, since the DAISY cohort was selected for having increased genetic risk of developing T1D. We did not measure intestinal permeability directly; instead we measured circulating IgG4 antibodies to dietary components in hope of observing an indirect measure of intestinal permeability. The DAISY study was not designed to specifically answer this research question, and the analysis may have been underpowered. However, the effect sizes of our negative findings were small. It is likely that, if a similar analysis conducted on a larger cohort found significant associations, they would be of modest effect size and unlikely to be clinically significant. Finally, we were unable to explore the presence of food allergies in the DAISY population for two reasons. First, DAISY collects parent-reported food allergies, but these reported allergies are not confirmed with clinical allergy testing. Second, we did not measure IgE antibody concentrations, as the measurement of food allergies was not the aim of this investigation.In conclusion, our results suggest that dietary IgG4 antibody concentrations in childhood may be related to timing of infant diet exposures. This study also demonstrates that IgG4 antibodies against common dietary antigens are relatively poor biomarkers of IA risk. We did find evidence that ovalbumin IgG4 antibody concentrations may be increased at IA development in children who subsequently develop T1D. This novel observation should be explored in other populations. Our examination of the individual dietary IgG4 antibody concentrations, as well as our composite measure of ß-lactoglobulin, gluten, and ovalbumin IgG4 antibodies in childhood, provided no evidence of a greater generalized immune response in children developing IA or progressing to T1D.


Information about antibiotic prescribing practice in primary care is not available for Ireland, unlike other European countries. The study aimed to ascertain the types of antibiotics and the corresponding conditions seen in primary care and whether general practitioners (GPs) felt that an antibiotic was necessary at the time of consultation. This information will be vital to inform future initiatives in prudent antibiotic prescribing in primary care.Participating GPs gathered data on all antibiotics prescribed by them in 100 consecutive patients’ consultations as well as data on the conditions being treated and whether they felt the antibiotic was necessary.nd and 3rd line agents being used for common ailments such as otitis media and tonsillitis. Amoxicillin, which is recommended as 1st line in most common infections, was twice as likely to be prescribed if the prescription was for deferred used or deemed unnecessary by the GP.171 GPs collected data on 16,899 consultations. An antibiotic was prescribed at 20.16% of these consultations. The majority were prescribed for symptoms or diagnoses associated with the respiratory system; the highest rate of prescribing in these consultations were for patients aged 15–64 years (62.23%). There is a high rate of 2The study demonstrates that potentially inappropriate prescribing is occurring in the adult population and the high rate of broad-spectrum antimicrobial agents is a major concern. This study also indicates that amoxicillin may be being used for its placebo effect rather than specifically for treatment of a definite bacterial infection. Antibiotic resistance is a major concern globally. Antibiotic consumption increases the likelihood for an individual to develop bacterial resistance . The majWide variation in antibiotic prescribing practices has been shown to exist in Europe demonstrating more than a threefold difference in antibiotic prescribing rates between countries, without any logical reasoning to explain the variation .E.coli increased from 5% in 2002 to 23% in 2008. Although ROI has seen small reductions in antibiotic use since 2009, high seasonal variation is still apparent which, in other countries, is also associated with high antibiotic consumption  [Streptococcus Pneumoniae (PNSP). The proportion of PNSP increased in ROI from 10.3% in 2004 to 23.1% in 2008 and decreased in 2010 to 18.2% [In the Republic of Ireland (ROI), there has been increasing levels of resistance and antibiotic use . Quinoloto 18.2% ,7.Non-clinical factors such as patient pressure have been shown to influence prescribing even in the absence of a clinical indication for an antibiotic ,9. The mThe purpose of this study was to describe specific diagnoses for which systemic antibiotics are prescribed in primary care by GPs in the ROI, to assess adherence to recent national guidelines and gain more information on antibiotics that are prescribed unnecessarily.The majority of GPs in ROI (> 80%) are regular attendees of the small group continuing medical education (CME) network which is run by the Irish College of General Practitioners (ICGP). The CME network is provided in a format whereby GPs meet on a regular basis to learn in a mutually supportive group. The system is resourced by 37 CME tutors nationally with a target population of 1,934 GPs and an attendance of over 80% of all GPs in Ireland  Tutors rAll CME tutors nationally and their CME groups were invited to participate in the study from October 2008 to April 2010. Each GP also completed an anonymous demographic questionnaire detailing their practice size, area, years in practice and post-graduate experience. All participating GPs were both state and privately funded. The state issues General Medical Service (GMS) cards to patients that are deemed unable to pay for medical care. Eligibility for GMS cards is dependent on a number of factors including income, marital status and age. In 2009, 33% of the population in Ireland were GMS card holders . All othParticipating GPs gathered data on 100 consecutive consultations using a predefined piloted data collection proforma  was provided by the Health Protection Surveillance Centre (HPSC). This data is from IMS Health, a pharmaceutical market research company and contains monthly wholesaler to retail pharmacy sales data from over 95% of the wholesalers and manufacturers in Ireland. Antibiotic consumption was measured in Defined Daily Dose (DDD) which is the assumed average maintenance dose per day for a drug used for its main indication in adults.Data was analysed using Microsoft Office Excel® (2007) and Predictive Analytics SoftWare  version 17.0. A sample (10%) was double-checked for coding entry errors. Data collected was tested for normality and parametric/non-parametric tests were used as appropriate. The Pearson’s chi-squared tests were performed to compare categorical variables. Odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were calculated using logistic regression.HPSC data was used to compare the pattern of antibiotic usage between the national picture and the study GP population.The study was approved by the Clinical Research Ethics Committee of the Cork Teaching Hospitals. Informed consent was obtained from all GPs by participation in the study.Data were collected from 171 GPs nationally who recorded information on 16,899 consultations. The mean (±SD) number of consultations recorded per GP was 98.82 ± 5.37. The majority of GPs who participated completed a demographics questionnaire . All GPs were from combined GMS and private practices and situated in various settings: urban , rural  and mixed The majority of attendees were females  and held a GMS card .An antibiotic was prescribed in one-fifth of consultations recorded . A quarter of these  were prescribed to a child between the ages of 1 and 14 years. A small proportion had received an antibiotic for the same presenting complaint in the previous two weeks prior to the consultation .Co-amoxiclav, amoxicillin and clarithromycin were the most commonly prescribed antibiotics and this was also reflected in the national data Table . With thSymptoms/diagnoses associated with the respiratory system accounted for the majority of antibiotic prescriptions , followed by skin  and urinary tract disorders . Overall, 22.63% of consultations  recorded either a diagnosis or symptoms of the respiratory system. The majority of these consultations received an antibiotic prescription . Children aged 4–14 years had the highest consultation rate where a respiratory symptom/diagnosis was recorded . Patients under the age of 65 were twice as likely to consult with respiratory symptoms/diagnoses than younger patients .Children aged from 0–14 years had the lowest percentage rate of antibiotic prescribing when presenting with respiratory symptoms 52.25%, 767). The highest percentage was seen for patients aged 15–64  . Nearly half of the deviations were related to the diagnosis/reason given for the consultation . This correlated with the high use of antibiotics for respiratory symptoms which are not included in the guidelines. The choice of the antibiotic was also a recurrent deviation . These included deviations such as the use of 2The majority of antibiotic prescriptions were for immediate use ; co-amoxiclav , amoxicillin  and clarithromycin  were the most common antibiotics prescribed. There were 470 (13.80%) antibiotic prescriptions for deferred use (2.05% (70) were not recorded). The majority of these prescriptions were amoxicillin , followed by co-amoxiclav  and phenoxymethylpenicillin . When a deferred prescription was issued, it was twice as likely to be for amoxicillin as any other antibiotic .Most of the antibiotics that were prescribed were deemed to be necessary for the condition they were being prescribed for  and almost a quarter of these prescriptions were for co-amoxiclav . There were 315 (9.25%) prescriptions that were thought not to be necessary and prescribers were unsure of 602 (17.67%) prescriptions; amoxicillin was the most common antibiotic choice in these instances  . This is higher than the UK where only approximately 15% of GP consultations are due to these ailments . As withThere is considerable variation in antibiotic prescribing between the respiratory clinical entities with a substantial proportion of prescribing occurring for respiratory symptoms. GPs may have had an inclination to label symptoms as a diagnosis to gain endorsement of their prescribing; diagnostic labelling has been shown to be associated with high levels of antibiotic prescribing .Antibiotics are not recommended for symptoms such as cough, sore throat but a high level of prescribing was recorded in these conditions. This is the reason why the majority of deviations from guidelines in this study were due to the diagnosis/reason for consultation. A study in Canada showed that 5% of URTI consultations received an antibiotic; this is in contrast to 33.10% in this study . The majIn comparison to other countries, the level of agreement to guidelines is low. However, these guidelines have been recently developed and should be disseminated widely to have any impact. In the Netherlands, about three-quarters of antibiotics prescribed are first-line choice . A thirdThe majority of tonsillitis presentations are not being treated with narrow-spectrum antibiotics as considered appropriate. This has a major impact on increasing resistance. The usage of amoxicillin for otitis media (first line) is lower than expected at 42.42%, other countries in Europe have rates of 82% with co-amoxiclav only used in 7% of cases . Studiesfollow the wish of the patient and avoid conflict’ [In contrast to the high rates of co-amoxiclav, amoxicillin was more likely to be prescribed than other antibiotics when the prescription was for deferred/delayed use and also when the GP did not think that the antibiotic was necessary for the condition being treated. It could be argued that this study provides possible evidence that GPs prescribe antibiotics such as amoxicillin as ‘impure placebos’ or ‘pseudoplacebos’, these agents consist of biologically active agents that have specific efficacy for some conditions but used as a placebo for another condition i.e. antibiotics for probable viral infections such as URTI and cough. A survey in Denmark found that 70% of GPs had prescribed antibiotics as a placebo intervention in the previous year . The mosonflict’ . Prescrionflict’ .This study clearly indicates that guidelines need to be disseminated at a local level with a focus to reduce antibiotic prescribing for minor respiratory conditions and reduce broad-spectrum antibiotic prescribing whenever necessary. Prescribing a course of antibiotics increases the patient’s risk of developing bacterial resistance for up to a year ; GPs shoThe data from this study is valuable as it is the first time diagnoses are linked to antibiotic consumption in ROI and also details prescribing behaviour for both private and GMS card holders. In ROI, private patients are not required to register with their GP and are therefore free to visit any GP they wish; patients do not have a unique identifier. Therefore, it is currently not possible for routine data collection to take place from electronic sources in ROI.The reason for the consultation was recorded in free text words to include all types of reasons provided for an antibiotic prescription. Other studies have quoted that a large proportion of primary care visits are difficult to label with a precise code and also require GPs to participate in training in coding . TherefoThe population of GPs in this study may be skewed towards those with an interest in the area of antibiotic prescribing as their participation was not mandatory as part of their CME group. However, the national data from the time of study reflects similar usage patterns of antibiotics. A possible explanation for the notable higher usage of doxycycline and lymecycline in the national dataset could be explained by dermatologists prescribing these agents in the outpatient setting.This study provides evidence that antibiotics are probably not being used as prudently in the community in ROI as they may be other countries. Children present to their GP with respiratory symptoms more often than adults, but higher rates of antibiotic prescribing occur in adults with similar symptoms. This study also shows that amoxicillin may be being used for its placebo effect rather than as specific treatment for bacterial infection.The authors declare that they have no conflict of interest.MM distributed the data collection booklets, analysed the data and drafted the original manuscript. All authors critically reviewed the manuscript and approved publication submission.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2296/13/43/prepubData Collection form.pdf. Description of data: This is a copy of the data collection form which participating GPs completed for the purposes of this study.Click here for file


In this study, we determined the impact of antibiotic homogeneity per se, and of cefepime versus antipseudomonal penicillin/β-lactamase inhibitor combinations (APP-β), on the likelihood of infection or colonisation with antibiotic resistant bacteria and/or two commonly resistant nosocomial pathogens (methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa). A secondary question was whether antibiotic cycling was associated with adverse outcomes including mortality, length of stay, and antibiotic resistance.Antibiotic homogeneity is thought to drive resistance but P. aeruginosa.We evaluated clinical and microbiological outcomes in two similar metropolitan ICUs, which both alternated cefepime with APP-β in four-month cycles. All microbiological isolates and commensal samples were analysed for the presence of antibiotic-resistant bacteria including MRSA and Length of stay, mortality and overall antibiotic resistance were unchanged after sixteen months. However, increased colonisation and infection by antibiotic-resistant bacteria were observed in cefepime cycles, returning to baseline in APP-β cycles. Cefepime was the strongest risk factor for acquisition of antibiotic-resistant infection.Ecological effects of different β-lactam antibiotics may be more important than specific activity against the causative agents or the effect of antibiotic homogeneity in selection for antibiotic resistance. This has important implications for antibiotic policy. Immediate effective antibiotic therapy is one of the most important intervention in severe sepsis, but ultimate survival is determined before cultures become positive and antibiotic susceptibilities available The clinical benefit of antibiotic-intensive strategies such as selective decontamination of the digestive tract P. aeruginosa  or of any bacteria resistant to cefepime, APP-β, or gentamicin, and considered these separately. A secondary aim was to determine whether the institution of antibiotic cycling in ICU was associated with adverse clinical outcomes, specifically relating to mortality and length of stay, in light of previous findings of increase mortality associated with empiric use of cefepime in the critically ill Our primary aim was to determine whether cycles of relatively homogeneous β-lactam use, alternating cefepime and APP-β as the core of empiric antibiotic therapy for sepsis in ICU, resulted in increased antibiotic resistance. We defined this as the acquisition at any site of either MRSA or We varied the β-lactam component (cefepime vs APP-β) of empiric antibiotic prescribing in four month cycles in two well-matched high-acuity metropolitan Australian ICUs. No attempt was made to influence prescribing for known or suspected aetiologies, but those judged in need of empiric broad-spectrum β-lactam therapy by the caring physician received cycle-specified β-lactam (cefepime or APP-β) with other antibiotics as deemed appropriate. Standard infection control procedures applied, according to matching guidelines in both institutions. Approval to conduct the study, under a waiver of consent, was obtained from the relevant Human Research Ethics Committees of the Sydney West Area Health Service and the Royal Brisbane and Women’s Hospital.There were four consecutive cycles in Unit 1 , beginning in April 2004 (cefepime), August (APP-β), December (cefepime), and April 2005 (APP-β), respectively , and thrOf the 1987 admissions to both ICUs in this period, 1194 were for at least 48 hours. Of these, 286 were admitted within 48 h of hospital arrival and received only cefepime or APP-β, or no antibiotics at all, by the end of their first week in ICU. In 206 of these (72%), late clinical followup and microbiological surveillance (see below) at all timepoints was complete. This group represents patients most clearly and directly subject to cycle-specific antibiotic influences. Data collected included age, sex, admission Acute Physiology and Chronic Health Evaluation (APACHE II) score, reason for admission, surgical and other interventions, ventilation and dialysis days, presence of central venous catheters, dialysis catheters, hospital and ICU length of stay (LOS), mortality, detailed antibiotic treatment, and microbiological information . The sta4 cfu/mL in non-bronchoscopic or directed bronchoalveloar lavage) or was predominant in a purulent sputum specimen without significant epithelial contamination evident  on microscopy. Isolates were also deemed significant if grown from normally sterile sites  or if predominant in the specimen on microscopy and culture  or if pyuria was present and >106 cfu/mL of a single organism was grown from urine. The reporting laboratory must also have issued full identification and susceptibility data on the basis of established standard laboratory criteria in both centres, in accordance with established definitions Positive cultures from all clinically indicated specimens submitted to the diagnostic laboratory were identified. Respiratory tract isolates were reported as significant if a pathogen was identified as sufficiently numerous in semi-quantitative assays (≥10Pseudomonas aeruginosa was confirmed by growth on Pseudomonas Isolation Agar (Becton Dickinson) Staphylococcus aureus (MRSA) by growth on Brilliance MRSA Agar (Oxoid) SmaI PFGE as previously described Enterobacteriaceae were detected by their morphology on chromogenic agar (CHROMagar Orientation) E. coli or of Klebsiella/Enterobacter/Serratia/Citrobacter spp. on CHROMAgar, in accordance with the Australian National Accreditation Testing Authority approved methodology in each laboratory and with the media manufacturer’s guidelines, were designated “Enterobacteriaceae” after testing to exclude oxidase activity. All clinical isolates of P. aeruginosa and all TIM-resistant P. aeruginosa isolates from surveillance samples, as well a random subset of TIM-resistant Enterobacteriaceae, were also tested against TZP. No significant differences were found in antimicrobial susceptibility so TIM was retained as the APP-β resistance marker. Unless otherwise specified in the text, ‘resistant’ or ‘resistance’ refers to APP-β, gentamicin and/or cefepime, in the above concentrations. All MRSA isolates were resistant to these three antibiotics, but P. aeruginosa isolates were specified as ‘resistant’ if resistant to any.All specimens were given unique numbers and de-identified before processing. Surveillance samples  obtained on admission and twice weekly thereafter were submitted to the centralised reference laboratories serving each unit (Unit 1: Centre for Infectious Diseases and Microbiology (CIDM) Laboratory Services at Westmead Hospital, Sydney; Unit 2: Queensland Pathology at the Royal Brisbane and Women’s Hospital, Brisbane), and inoculated into nutrient broth overnight, resuspended in 20% glycerol in nutrient broth  and stored at -80°C for transfer to CIDM. A loopful of each frozen sample was resuspended in nutrient broth at 37°C for 90 min aerobically before inoculation onto solid media. P. aeruginosa and methicillin-resistant S. aureus was the primary outcome for analysis. Each significant isolate and each antibiotic-resistant colonisation was counted only once per admission. Data were analysed separately for both hospitals before being pooled, as the differences between the two Units in terms of casemix and basic demographics were minor (see Acquisition of infection and/or colonisation by antibiotic-resistant bacteria and by inor see . StatistAdmissions to Unit 1 (n = 1135) and Unit 2 (n = 852) were similar in casemix and other key characteristics , and werAntibiotic homogeneity was high: cycle-specified drug comprised >60% of major β-lactam antibiotic use in respective on-cycles and <15% in off-cycles . Other aP. aeruginosa or other antibiotic resistant bacteria. Two hundred and twenty-three admissions in all were complicated at some stage by resistant infection. Of all infecting bacteria resistant to any of the three first-line antibiotics, APP-β  and gentamicin resistance  were most common. Cefepime resistance was rare among Enterobacteriaceae  and P. aeruginosa . A cefepime-resistant infection complicated only 32 (14.3%) admissions. Forty-one (18.4%) of the 223 admissions complicated by resistant infection were complicated by antibiotic-resistant P. aeruginosa and 81/223 (36.3%) by MRSA.In order to determine the impact of the two different β-lactam regimens, we compared the risk of infection and/or colonisation with either MRSA or P. aeruginosa infection  complicated a greater proportion of admissions in cefepime than APP-β cycles . MRSA infection complicated significantly more cefepime than APP-β cycle admissions , and this was also evident between individual cycles . P. aeruβ cycles , and thiEnterobacteriaceae but few were antibiotic-resistant and these were not further analysed.Fourteen cefepime-cycle admissions were complicated by antibiotic-resistant bacteraemia compared to nine APP-β-cycle admissions although the difference between these small numbers was not significant . About a third of all bacteraemias  were due to P. aeruginosa was isolated from surveillance samples (Increased infection in those exposed to cefepime should be associated with increased colonisation rates. We identified admissions direct from the community who received either specified β-lactam (and no off-cycle β-lactam) for >48 hours before sampling in the first week of admission, or no antibiotics at all . This subset (n = 286), with higher admission APACHE II scores and ICU length of stay , most ac samples . However samples , and man samples .P. aeruginosa and 44 (69.8%) acquired resistant Enterobacteriaceae. Backwards stepwise logistic regression analysis revealed cefepime treatment in a cefepime cycle as the sole independent predictor of acquiring resistant bacteria at surveillance sites . Those treated with cefepime were significantly more likely to acquire MRSA  or antibiotic-resistant Enterobacteriaceae . New acquisition of (any) P. aeruginosa was also twice as common in the cefepime than APP-β -treated admissions, but this was not quite statistically significant at 95% confidence levels . The only factor associated with reduced risk of antibiotic-resistant colonization was an operative intervention . Although APP-β treatment did not independently predict reduced risk, cefepime treatment is independently predictive of increased risk of resistance acquisition in multivariate analysis.Sampling was complete at all time points in 206 of these 286 admissions. Of these 206, 63 (30.6%) developed perineal and/or endotracheal colonisation by antibiotic-resistant bacteria in the first week. Of 63 newly colonised admissions identified, 18 (28.6%) acquired MRSA, 36 (57.1%) acquired Clonal MRSA outbreaks that were coincidental with the antibiotic cycle changes were excluded as an explanation for these findings. Standardised methodology The model of antibiotic cycling is part of normal ICU practice, and in similar studies in vitro but APP-β seem to have less adverse effects on the microflora than third-generation cephalosporins Enterobacteriaceae with class 1 cephalosporinases is dependent on the tazobactam component, which is relatively less excreted in bile than piperacillin in vitro activity of piperacillin is superior to that of cefepime against the major anaerobic pathogens Clostridum difficileP. aeruginosaEnterobacteriaceaeP. aeruginosa infection rates also rose and fell according to cycle, and underlying colonisation rates appeared to behave in the same way.We found no support for mathematical models that identify antibiotic homogeneity as a specific resistance driver P. aeruginosa colonisation and infection, and significantly so for combined FEP cycles (27/362) vs APP-β cycles (14/381) in Unit 1 (p 0.024). Ratios between bacteraemia and respiratory infection and deep isolates were similar between Units for MRSA and for methicillin-sensitive S. aureus (MSSA), although respiratory infection was more commonly reported in Unit 2 in general, and this included Gram-negative pathogens as well as S. aureus  may have arisen indirectly, through greater awareness of the pathogen, and this would be another potential confounder. Any bias introduced by relatively higher incidence of community admissions to Unit 2 introduces would be expected to operate to reduce rates of nosocomial Pseudomonas infection but Unit 2 also reported a higher rate of urinary catheterization which, combined with the possible variation in respiratory tract reporting, may relatively increase apparent Pseudomonas infection rates. In any case however, the fact that infection was less common in Unit 2 than Unit 1 in almost every site including the bloodstream and that only Unit 2 experienced P. aeruginosa bacteraemias suggests that the difference is real. This is difficult to further clarify, although it is reasonable to assume that any attribution of undue significance to respiratory P. aeruginosa in Unit 2 and/or under-attribution in Unit 1 would be unaffected by cycling and remain relatively constant from cycle to cycle.Cefepime was associated with increased . aureus . Indeed,Neither length of stay nor mortality changed significantly overall in this study, despite the known consequences of antibiotic-resistant infection per se does not appear to be a specific resistance driver. The ecological influences of cefepime seem to exceed that of all other risk factors in determining infection risk and may be relevant to unexplained mortality differences between cefepime and TZP in large studies of the critically ill. Failure to consider ecological effects in setting antibiotic policy may increase antibiotic resistance and even contribute to unintended mortality.Our data show that cefepime therapy is associated with increased infection due to organisms resistant to the key antibiotics used for management of sepsis and septic shock in intensive care. Importantly, prescribing homogeneity Figure S1Temporal distribution of MRSA pulsotypes within cycling period. ST: Sequence type (letters indicate subgroups within STs at 95% identity). FEP: cefepime cycles; APP-β: antipseudomonal penicillin combination cycles.(TIF)Click here for additional data file.Figure S2Monthly MRSA acquisition rates in Unit 1 before, during and after cycling , shown as patients positive per 100 bed days. FEP: cefepime cycles; APP-β: antipseudomonal penicillin combination cycle.(TIF)Click here for additional data file.Table S1Patient characteristics of all admissions in which sampling was complete (n = 206).aMann-Whitney U test or Chi-Squared analysis.(DOC)Click here for additional data file.


In influenza, the envelope protein hemagglutinin (HA) plays a critical role in viral entry by first binding to sialic acid receptors on the cell surface and subsequently mediating fusion of the viral and target membranes. In this work, the receptor binding properties of influenza A HA from different subtypes  have been characterized by NMR spectroscopy. Using saturation transfer difference (STD) NMR, we find that all HAs bind to the receptor analogs 2,3-sialyllactose and 2,6-sialyllactose, with subtle differences in the binding mode. Using competition STD NMR, we determine the receptor preferences for the HA subtypes. We find that H5-Qinghai and H9-Hong Kong HA bind to both receptor analogs with similar affinity. On the other hand, H1 exhibits a clear preference for 2,6-sialyllactose while H5-Vietnam exhibits a clear preference for 2,3-sialyllactose. Together, these results are interpreted within the context of differences in both the amino acid sequence and structures of HA from the different subtypes in determining receptor preference. The membrane glycoproteins hemagglutinin (HA) and neuraminidase (NA) play critical roles in influenza infection HA, as well as analogous envelope proteins from Ebola, HIV, and sialic acidRS-CoV, mediates virus entry through binding to receptor and conformational changes that result in fusion of the viral and target cell membranes Due to its critical nature, the initial step of influenza entry, HA binding to sialic acid, has received much attention 1H are selectively irradiated (HA in this case), and subsequently magnetization is transferred to the 1H of small molecules (the SL) that exchange between bound and free states during the irradiation period; the difference, with respect to a reference spectrum in which the large molecule 1H are not irradiated and hence no magnetization transfer occurs, identifies 1H of the small molecule that are in closest contact to the large molecule in the bound state 3eq1H resonates as a doublet at 2.73 ppm and the 6′SL H3eq1H resonates as a doublet at 2.67 ppm and thus 3′SL and 6′SL binding are readily determined by the H3eq resonance. From d of ∼3 mM In this work we characterize the receptor binding properties of the HA from three different subtypes of influenza A: H1, H5 and H9. The H1 HA was from a human source of the recent 2009 outbreak, which represents the first pandemic influenza outbreak of the 21st century. The H5 HA were from a human source, H5 HA/Vietnam (hereafter referred to as H5-V), and an avian source, H5 HA/Qinghai (hereafter referred to as H5-Q), both of which represent highly pathogenic H5 isolates. The H9 HA was from a human source of the 1999 outbreak. We chose to use 2,3-siallylactose 3′SL,  and 2,6-1H within a particular ligand gives insight into their proximity to the protein interaction surface 3eq (discussed above), H3ax, and the N-acetyl group ∧2+(NIoff/Ioff)∧2)∧0.5 ΔI and NIoff are the noise calculated by NMRDraw in the appropriate spectrum. We note that the STD experiment can be used to determine Kd in certain cases d by titration failed due to additional binding of sialic acid at concentrations greater than 5 mM, as manifested by an inability to achieve ligand saturation.The HA samples from subtypes H1 A/California/04/09 (H1N1), H5 A/bar-headed goose/Qinghai/1A/05 (H5N1), H5 A/Vietnam/1205/04 (H5N1), and H9 A/Hong Kong/1073/99 (H9N2) were obtained from BEI Resources . In each case the HA are full length, prepared in cell culture, and glycosylated. The purity of each HA was verified by SDS-PAGE. The sialic acid receptor analogs, 2,3-sialyllactose (3′SL) and 2,6-sialyllactose (6′SL), were obtained from V-Labs . For the STD experiments, the experimental conditions were 3 mM sialic acid and 2 µM (monomer) of the respective HA in PBS buffer (pH 7.4). STD NMR experiments were performed with a train of 50 msec gaussian-shaped saturating pulses at 100 Hz power for 3 sec with “on” resonance saturation at −0.4 ppm and “off” resonance saturation at 30 ppm (the relaxation delay was 2 sec before the saturating pulses). The number of scans was 1728 and the spectral width was 14,367 Hz. Spectra were recorded at 25°C on a Bruker AVANCE 900 MHz spectrometer equipped with a cryogenic probe. Spectra were processed by NMRPipe with a 5 Hz line broadening and analyzed by NMRDraw Figure S1STD NMR spectrum of 3′SL and 6′SL in the absence of HA (the control experiment for the STD). The experimental conditions were 3 mM SA in PBS (pH 7.4) at 25°C. Note that these spectra were obtained on a Bruker 800 MHz spectrometer equipped with a room temperature triple resonance probe.(PDF)Click here for additional data file.Figure S2STD competition assay for H5-Q binding to 3′SL and 6′SL. The experimental conditions were 2 uM HA, 3 mM 6′SL, 3 mM 3′SL (upper spectrum) or 6 mM 3′SL (lower spectrum) in PBS, pH 7.4 at 25°C.(PDF)Click here for additional data file.Figure S3Structural alignment of influenza HA H1 (red) and H5-V (blue) backbones. The green residues correspond to residues that interact with SA.(PDF)Click here for additional data file.Table S11.% STD intensity for HA-SL interactions(PDF)Click here for additional data file.Materials and Methods S1(PDF)Click here for additional data file.


The viable but nonculturable (VBNC) state is a unique survival strategy of many bacteria in the environment in response to adverse environmental conditions. VBNC bacteria cannot be cultured on routine microbiological media, but they remain viable and retain virulence. The VBNC bacteria can be resuscitated when provided with appropriate conditions. A good number of bacteria including many human pathogens have been reported to enter the VBNC state. Though there have been disputes on the existence of VBNC in the past, extensive molecular studies have resolved most of them, and VBNC has been accepted as a distinct survival state. VBNC pathogenic bacteria are considered a threat to public health and food safety due to their nondetectability through conventional food and water testing methods. A number of disease outbreaks have been reported where VBNC bacteria have been implicated as the causative agent. Further molecular and combinatorial research is needed to tackle the threat posed by VBNC bacteria with regard to public health and food safety. The cells that form a colony on specific nutrient media are the culturable cells. Viable means metabolically or physiologically active. So the cells that are metabolically or physiologically active but cannot be cultured on specific media are the viable but nonculturable cells (VBNC) . Most miRhizobium, Agrobacterium, and Helicobacter-Campylobacter species [In 1982, Xu and coworkers introduced the term “viable but nonculturable bacterial cells (VBNC)” to distinguish particular cells that could not form colonies on solid media but retained metabolic activity and the ability to elongate after the administration of nutrients . Accordi species .Vibrio cholerae from streams and drainage ditches, including sites with negligible chance of sewage contamination. Around the same time, Colwell et al. [Vibrio cholerae in Maryland, USA. She and her coworkers showed that, in artificial sea water, Vibrio cholera and E. coli remained viable though they lost the capacity to form colonies on culture media [Salmonella enteritidis, Shigella sonnei, and Legionella pneumophila joined the list of organisms known to be capable of entering a state in which they failed to show up on nutrient agar yet took up substrates and signaled in other ways that they were certainly not dead [Bashford and colleagues  announcel et al.  also foure media . Soon Sanot dead . The usenot dead .Vibrio cholerae, enteropathogenic E. coli, Legionella pneumophila, and various other bacteria have been shown to regain culturability after they have entered the intestinal tracts of animals [Microorganisms that do not grow in culture media, but are still metabolically active and capable of causing infections in animals and plants, are said to be in a VBNC state. Traditional laboratory culture conditions and methods cannot meet the requirements of VBNC organisms to resume growth . Semista animals .sok/hak or autolysins [The VBNC state is defined as a state of dormancy triggered by harsh environmental conditions , such astolysins ; and dectolysins  and chlotolysins .maintaining apparent cell integrity; possessing some form of measurable cellular activity ;in vivo [possessing apparent capacity to regain culturability in vivo ; responding to external stimulus as shown by specific gene expression ; having low metabolic activity ; exhibiting dwarfing ; having reduced nutrient transport; containing a high ATP level and exhibit high membrane potential ; having extensive modifications to the fatty acid composition in cytoplasmic membranes ; within the cell wall peptidoglycan, increased crosslinking, increased muropeptides bearing covalently bound lipoprotein, and shortening of the average length of glycan strands ; higher autolytic capability than exponentially growing cells; retained plasmids; increased antibiotic resistance due to lower metabolic activity ; changes in outer-membrane protein profile ; continuous gene expression .The VBNC state continues to be disputed due to the difficulties of differentiating VBNC cells and dormant cells through resuscitation and phenotypic studies. However, recent molecular studies have generated data to support the existence of the VBNC state [The characteristics of bacteria in the VBNC state can be summarized as follows:NC state .The VBNC is also considered an important reservoir of many human pathogens in the environment .Aeromonas hydrophila, Agrobacterium tumefaciens, Burkholderia cepacia, Campylobacter jejuni, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecalis, Escherichia coli (including EHEC), Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Listeria monocytogenes, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Salmonella typhi, Salmonella typhimurium, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Streptococcus faecalis, Vibrio alginolyticus, Vibrio cholerae, Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio vulnificus (types 1 and 2).The following list includes but is not limited to the pathogenic bacteria that can enter the VBNC state : AeromonVibrio cholerae O1, followed by human volunteer experiments [E. coli nonculturable cells were also reisolated after passaging through rabbit ileal loops 4 days after inoculation, and chick embryos died when injected with nonculturable cells of Legionella pneumophila, leading to the conclusion that VBNC pathogens remain potentially pathogenic. So, VBNC has a huge significance in public health care [Many believe that pathogens in the VBNC state are unable to induce infection/disease despite retaining their virulent properties. However, when VBNC pathogens pass through a host animal , resuscieriments . VBNC E.lth care .Many indicator bacteria and pathogenic bacteria which exist in aquatic habitats have been shown to have a VBNC state . Water iVibrio cholerae O1 in the surface water have been shown to remain in nonculturable state. These water sources are used for domestic purpose regularly and pose a risk of infection [Vibrio cholerae in water in the VBNC state is an important public health factor, since detection will not be successful if only conventional cultural methods are used [Thus, food and environmental and clinical samples no longer can be considered free from pathogens if culturing yields negative results. For the general public, the presence of VBNC in water and food may be related to low-grade infections or the so-called aseptic infections. In many cases, the infections are incorrectly attributed to viruses since no bacteria were detected. For example, nfection . When coare used .Shigella can undergo VBNC state in water but become a threat when they enter the human body [E. coli cells which remained in VBNC state [E. coli retain enteropathogenicity as shown by continual production of enterotoxin [Helicobacter pylori cells can express virulence factors such as cagA, vacA, and vreA.Similarly, man body . Additioman body . One stuNC state . FurtherNC state . Other serotoxin . Nilssonerotoxin  showed taw, and chemical composition) and environmental factors  act simultaneously on contaminating bacteria leading to the VNBC state [E. coli O157 : H7 and S. typhimurium within 24 hours of incubation [E. coli and Pseudomonas putida enter into the VBNC state but retain transcription and translation functions. Several foodborne outbreaks have been reported in Japan, where pathogens such as Salmonella enterica subsp. enterica [E. coli O157 [The presence of VBNC bacteria in food is well documented . Food anBC state . For exacubation . Again, cubation  reportedoli O157  in the VThe VNBC is also critical in determining shelf life and microbial stability of food and beverages. For example, acetic acid and lactic acid bacteria entered the VBNC state in wine as a consequence of lack of oxygen and presence of sulphites .Nalidixic acid (20–40 mg/L) is used to stop cell division. After exposure to nalidixic acid, viable cells continue to grow and will appear elongated, whereas the nonviable metabolically inactive cells will retain their original shape and size. The cells are then observed under a microscope. Viable cells will be seen as elongated, whereas VBNC/dormant cells will be seen as oval and large .Various fluorescent staining procedures can be used to determine VBNC organisms. Frequently used stains are acridine orange, 4,6-diamino-2-phenyl indole (DAPI), fluorescein isothiocyanate (FITC), indophenyl-nitrophenyl-phenyltetrazolium chloride (INT), and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) . The modBacLight Live/Dead assay, has been developed. The assay allows simultaneous counting total and viable  cells, by using two nucleic acid stains, that is, green-fluorescent SYTO 9 stain and red-fluorescent propidium iodide stain. SYTO 9 stains both live and dead bacteria, whereas propidium iodide penetrates only bacteria having damaged membranes. When used together, propidium iodide reduces SYTO 9 fluorescence in dead bacteria with damaged membranes resulting in red fluorescent cells, whereas the live bacteria will fluoresce green [In recent years, a new differential staining assay, the ce green . Hybridization probes are nucleic acids (DNA/RNA) which have been chemically or radioactively labeled and are used to detect complementary target DNA/RNA. Specific amplification of DNA targets in bulk DNA extracts from environmental and clinical samples permits detection of specific organisms or groups of related organisms without the need to cultivate them, provided the appropriate unique primers are used . These pEven though traditional culture methods fail to detect the presence of specific VBNC in a sample, the presence of these microbes can be demonstrated using some of the molecular techniques described. More specifically, oligonucleotide probes of l8–20 nucleotides are proving most useful because they hybridize rapidly to specific DNA sequences of target organisms. These gene probes can reveal closely related organisms or organisms with similar functional capabilities. Additional molecular techniques are then required to fully identify any VNBC detected . The detFrom the above discussions, it is evident that a number of nonspore-forming human pathogenic bacteria can enter the VBNC state with maintained cellular structure and biology and persistent gene expression but remain nonculturable by traditional cultural techniques. They can survive and revert to culturable conditions when provided with appropriate conditions, hence, being a significant threat to public health and food safety. Further research is needed to elucidate mechanisms leading to the VBNC and the development of methodologies to confirm their existence.


Over the past decade and with increasing pace in the last few years, a “new” disease has emerged, gradually affecting a wide range of medical specialties and explaining a host of conditions previously regarded as separate entities. This newly recognized condition is IgG4-related disease (IgG4-RD), a potentially multiorgan disorder that is characterized by elevated serum IgG4 concentrations in the majority of cases. IgG4-RD was recognized in modern times in Japan through a series of seminal observations that occurred during the 1990s and the first few years of this century , but it In addition to the frequent elevations of serum IgG4 concentrations, certain major pathologic hallmarks are generally present to one degree or another across all organ systems, providing the principal foundation for the belief that the disparate organ manifestations associated with this diagnosis are in fact part of the same systemic disease. These pathologic features include a lymphoplasmacytic infiltrate with a high percentage of plasma cells within the lesion staining for IgG4; a peculiar pattern of fibrosis known as “storiform” fibrosis; a tendency to affect veins in a manner that leads to obliterative phlebitis; and mild to moderate tissue eosinophilia . IgG4-RD appears to sit at an intersection between different inflammatory pathways. Many but not all patients have substantial allergic or atopic histories, and early indications are that a “modified” Th2 response is critical to this condition . Other pIgG4-RD is now recognized as a worldwide disease . The intIn this special issue, we are pleased to present more than two dozen papers on IgG4-RD that address a number of facets of this condition: from its clinical manifestations to its radiologic features; from its pathology hallmarks to its serologic characteristics; and from its diagnostic challenges to early indications of treatment success. These papers capture the essence of IgG4-RD in 2012 and represent the current state-of-the-art against which future advances will be compared.John H. StoneJohn H. StoneJohn K. C. ChanJohn K. C. ChanVikram DeshpandeVikram DeshpandeKazuichi OkazakiKazuichi OkazakiHisanori UmeharaHisanori UmeharaYoh ZenYoh Zen


The application of nanotechnology in biology and medicine represents a significant paradigm shift in the approach to the treatment of cancer. Evidence suggests that when exposed to near-infrared radiation (NIR), carbon nanotubes (CNTs) dissipate a substantial amount of heat energy. We have developed a novel nanocomposite polymer, polyhedral oligomeric silsesquioxane poly (carbonate-urea) urethane (POSS-PCU). POSS-PCU displays excellent biocompatibility and has been used in making artificial organs as well as protective coatings for medical devices.Functionalizing (or “coating”) CNTs with POSS-PCU confers biocompatibility and increase the amount of heat energy generated, by enhancing dispersion. Here we demonstrate that POSS-PCU-functionalized multi-walled CNTs (MWNTs) act synergistically together when exposed to NIR to thermally ablate cancer cells.Given that POSS-PCU has already been used in human in first-in-man studies as trachea, lacrimal duct, bypass graft and other organs, our long-term goal is to take POSS-PCU coated CNTs to clinical studies to address the treatment of cancer by optimizing its therapeutic index and increasing its specificity via antibody conjugation. Carbon nanotubes (CNTs) were initially described as “helical microtubules of graphitic carbon” by Sumio Ijima (of NEC Corporation), in a letter to Nature in 1991 , to ther2 hybridized carbon atoms, while MWNTs are concentric layers of SWNTs [Generally, CNTs can be classified into two broad categories: multi-walled carbon nanotubes (MWNTs) and single-walled carbon nanotubes (SWNTs). SWNTs can be conceptualized as a rolled-up sheet of planar spof SWNTs . CNTs haof SWNTs   urethane . POSS-PCU is biocompatible , resistaA major concern for the use of CNTs in biological systems is its insolubility and inherent toxicity. With proper functionalization, for example with polyethylene glycol (PEG), CNTs appear to be well-tolerated in animal studies . Hence, Raw CNTs appear as a black powder-like substance, which is insoluble in water. Upon functionalization with POSS-PCU via bath sonication, the final product appears as a homogenous mixture Figure  suitableRaw CNTs are insoluble in water and biological systems. Upon non-covalent functionalization with POSS-PCU, solubility was conferred, rendering it ready for use in biological systems. POSS-PCU is highly compatible, and its use in humans as a bioartificial trachea, bypass graft and lacrimal duct underscores its potential usability as a coating agent for nanoparticles as well. Unfunctionalized CNTs are not appropriate for use in biological systems, and we postulate that biocompatibility can be conferred via functionalization with POSS-PCU.As POSS-PCU has thermoplastic behavior, does not absorb in the near infrared spectrum. Thus, when exposed to NIR laser, the temperature of POSS-PCU remains constant at room temperature. Our results reveal that the presence of POSS-PCU does not simply maintain the temperature profile curve (as one might expect due to its absence of heating effect), but instead increases the temperature profile curve when functionalized with CNTs.Upon functionalization with POSS-PCU, CNTs were able to attain a greater rate of temperature increase as well as a higher final temperature of 88°C within ten minutes of NIR laser exposure compared to non-functionalized CNTs p = 0.0006). At both wattages, synergistic thermal effects were observed, as POSS-PCU-CNT was able to attain a higher temperature than POSS-PCU and CNTs alone , possibly due to the inherent toxicity of CNTs. In contrast, treatment with POSS-PCU-CNT (without laser) did not result in any significant change in cell number, indicating that functionalization with POSS-PCU had conferred a high degree of biocompatibility to CNTs. Concurrent with established literature, a decrease in cell number (of around 50%) was observed when  CNTs were irradiated with NIR laser. More importantly, when POSS-PCU-CNT was irradiated with NIR laser, there was a dramatic decrease in cell numbers, thermally ablating 95% of cancer cells (p = 0.0000842)  were obtained from Nanothinx S.A. . They had a mean diameter of 15 nm, and a mean length of 100 nm. The preparation of POSS-PCU has been described elsewhere. BrieflyVisual characterization was conducted using a JEM-2100 F transmission electron microscope (JEOL Ltd. UK). Optical absorbance of CNT, POSS-PCU, and POSS-PCU-CNT was obtained using a LAMBDA 1050 UV/Vis/NIR . Scan parameters were as follows: start (4000), number of scans (100), resolution (4), units (%T), end (400), interval (4), shuttle (interleaved). The absorption spectrum was obtained via a second derivative and a 16-point reading.A NIR laser system  with a 808 nm laser diode was used to irradiate CNTs. The laser diode controller was set to either 2300 mA (1 W) or 1250 mA (0.5 W). To prevent over-heating, the temperature controller was set to 10 kΩ. Temperature tracking and thermal images were obtained using a Thermacam™ SC500 thermal camera . CNTs were exposed to NIR irradiation for 10 minutes, and temperature changes were recorded every minute.6) cells were counted and seeded on a 96-well plate, with a theoretical cell count of 300,000 in each well. To each well, 100 μl of treatment reagent was added . All treatments were done in triplicates. NIR laser was then applied for 10 minutes. Cells were then incubated at 37°C with 5% CO2 for 24 hours, and counted thereafter. All reagents pertaining to cell work were purchased from Invitrogen. Cell count was done using a MACSQuant Analyzer flow cytometry .A HT-29 human colorectal cancer cell line was used as a model for photothermal cancer ablation. Briefly, 3 million  and statistical analyses were conducted at a 95% confidence interval using MATLAB® . Statistical significance testing was conducted using unpaired Student’s The authors declare that they have not competing interests.AT, SYM, JR, GP planned, carried out, and wrote the manuscript. AMS conceived the study, participated in the design and coordination, and helped draft the manuscript. All authors read and approved the final manuscript.