The SCPE score measures the distance between the trial sequence and a given reference sequence, given the structure. In our first brief report we used a "global score", in which the same reference sequence, the ancestral one, was used at each evolutionary step. Here, we introduce a new scoring function, the "local score", in which the sequence accepted at the previous evolutionary time-step is used as the reference. We assess the model on the UDP-N-acetylglucosamine acyltransferase (LPXA) family, as in our previous report, and we extend this study to all other members of the left-handed parallel beta helix fold (LβH) superfamily whose structure has been determined.Structure conservation constrains evolutionary sequence divergence, resulting in observable sequence patterns. Most current models of protein evolution do not take structure into account explicitly, being unsuitable for investigating the effects of structure conservation on sequence divergence. To this end, we recently developed the Structurally Constrained Protein Evolution (SCPE) model. The model starts with the coding sequence of a protein with known three-dimensional structure. At each evolutionary time-step of an SCPE simulation, a trial sequence is generated by introducing a random point mutation in the current coding DNA sequence. Then, a "score" for the trial sequence is calculated and the mutation is accepted only if its score is under a given cutoff, i of other members within the LβH superfamily.We studied site-dependent entropies, amino acid probability distributions, and substitution matrices predicted by SCPE and compared with experimental data for several members of the LβH superfamily. We also evaluated structure conservation during simulations. Overall, SCPE outperforms JTT in the description of sequence patterns observed in structurally constrained sites. Maximum Likelihood calculations show that the local-score and global-score SCPE substitution matrices obtained for LPXA outperform the JTT model for the LPXA family and for the structurally constrained sites of class We extended the SCPE model by introducing a new scoring function, the local score. We performed a thorough assessment of the SCPE model on the LPXA family and extended it to all other members of known structure of the LβH superfamily. Protein structure is more conserved than protein sequence during molecular evolution -3. RemotThe restrictions imposed by the environment of a given protein site onto its pattern of amino acid substitutions have been largely discussed ,5-9. Briλ, that measures the amount of structural perturbation allowed by natural selection. In this way, for λ = 0 only synonymous mutations are accepted, whereas for λ ~ ∞ all mutations are accepted. The procedure is repeated until a desired number of mutations are reached. In the present work the DNA is mutated using the Jukes-Cantor model, so that each nucleotide substitution occurs with the same probability.To study how protein structure conservation modulates sequence divergence, we recently developed the Structurally Constrained Protein Evolution (SCPE) model . The staλ, that must be fit by comparison to actual sequence data. Different properties could be used to fit the cut-off. As we will show below, the model is quite robust with respect to the property used. Therefore, we have used the simplest way, which is to fit λ such that the acceptance rate, ω, inferred for actual sequences is reproduced. The acceptance rate is the probability that an amino acid mutation is accepted. Thus, it can be estimated by the ratio between the number of amino acid substitutions (accepted mutations) and the total number of trial amino acid mutations. The acceptance rate has been extensively used to characterize the strength of the selective pressure under which proteins evolve [ω would be 1. In general the ω values are usually below 0.5 due to the deleterious effects of most amino acid mutations [ω can take values very close to zero.The model depends on one parameter, the cut-off s evolve -27. If autations . In protOne of the main factors determining the quality of the SCPE model is the scoring function. Given the structure of the ancestral protein, which we assume constant throughout the simulation, the score of a given trial sequence is defined as the RMSD between the mean-field energy profile of the trial sequence and that of a reference sequence. In our previous work, the same reference sequence, the ancestral one, was used for each time-step. Therefore, the score of each trial sequence was a measure of the dissimilarity between the trial sequence and the ancestral sequence, given the structure. Such a score depends only on the trial sequence and the ancestral sequence, but not on the particular sequence mutated to obtain the trial. Hence, it does not depend on the precise evolutionary path between the ancestral and the trial. Therefore, this will be called from now on "global score".Even though the global score has been proved to be very good at reproducing the sequence patterns of a test case, it also shows some problems. Mainly, at the beginning of a simulation most mutations fall below the optimum cut-off. This results in too high values of the acceptance rate. Only after about 5% of the sites have been substituted, the cut-off is purifying enough to reproduce the acceptance rate inferred for the actual family. From a more qualitative point of view, since at the beginning of global-score simulations almost all mutations are accepted, erroneous amino acids, which are not found in the natural sequences of the family studied, can be introduced with relatively high probability during the first few steps of a global-score simulation. We shall see below that these are unwanted artefacts of the global-score SCPE simulations.To tackle the problems described in the previous paragraph, in this paper we introduce a "local score", in which the reference sequence for a given trial is that accepted in the previous evolutionary time step, the current sequence, rather than the ancestral one. Thus, the local score measures the mutational perturbation introduced in a given time-step, rather than the global difference between the trial and ancestral sequences.Escherichia coli. A portion of this protein presents a left-handed parallel beta helix (LβH), a fold generally associated with transferase activity and broadly distributed in different taxons [The new approach is compared with the previous one on the same test system studied before: UDP-N-acetylglucosamine acyltransferase (LPXA) from t taxons -31 .In Figure λ so that the acceptance rate of the simulations matches that inferred from actual sequences. For the SCPE simulations, the value of ω is easily estimated by just counting the number of amino acid substitutions accepted throughout the simulations and dividing by the number of trial amino acid mutations. For the reference alignment, however, one has to estimate ω using some inference method. These methods tend to overestimate the actual ω. This can be seen in Figure ω inferences for a set of SCPE simulations as a function of the cut-off λ, together with the value calculated by counting the proportion of accepted mutations (see Methods). The inferences were made with the module yn00 of PAML [ω departs from the calculated ω as λ increases. This behaviour is expected since, for a given number of mutations, for larger λ there are more accepted nonsynonymous substitutions, which results in loss of sequence signal.As discussed in Methods, we have chosen to determine the optimum value of the SCPE model parameter  of PAML  as explaω, we obtained the ω of the reference alignment of 25 sequences homologous to the UDP-N-acetylglucosamine acyltransferase from Escherichia coli . The average ω for this alignment is 0.22. Using this value in Figure λ obtained are 1.10 and 7.00 for local and global score, respectively.Using the method yn00+w+f, which best estimates the calculated λ values for local and global score are very different. Thus, for the sake of comparison, we take advantage of the one-to-one relationship between λ and ω, shown in Figures ω instead of λ as model parameter. In Figure ω versus the calculated ω for local-score and global-score simulations. Using this plot and the inferred ω value for the LPXA reference alignment, ω = 0.22 ± 0.11 (0.11 is the standard deviation of ω), we calculate an optimal ω of 0.15 (0.12–0.27) for local score and 0.19 (0.12–0.26) for global score.We note here that the optimal It is important to assess if the SCPE models are able to preserve protein structure. To this end we used THREADER 3 to analyze the percentage of sequences that recognize the correct structure using different models. Results are shown in Table a priori, that in the long term the global-score would be better at conserving structure than the local-score, since in the later case the reference sequence is reset at each step so that it would be easier to lose memory of the ancestral protein. One of the reasons of the global-score SCPE being worse at conserving structure could be the erroneous amino acid substitutions introduced at the beginning of the simulations (see above). To gain more insight into this issue, further work involving much longer simulations would be needed. However, for long enough evolutionary time it is not longer reasonable to assume that structure remains constant. In this limit, any model based on assuming structural conservation will break down.When both SCPE schemes are compared, Table i and i+4.To evaluate the capacity of the SCPE model to reproduce the sequence patterns found in the LPXA family, the variability of each site was analysed. The different protein positions were accumulated into 6 structural classes. For each class, we calculated the entropies corresponding to the equilibrium distributions of SCPE models. These entropies represent the average structural constraints of each structural class and do not depend on simulation time. SCPE entropy profiles are compared with those obtained from the reference alignment of the LPXA family. One could argue that these are not only determined by structure, but also contain historical information. However, since we are accumulating over several sites of the same class, which would have independent evolutionary histories, we expect such information to be somewhat averaged out. For the sake of comparison we also calculated the entropy profiles of JTT simulations of 0.28 amino acid substitutions per site (see Methods). The resulting entropy profiles are shown in Figure ω is shown. Comparing Figures ω for which the entropy error is minimum is consistent with the value at the optimum cut-off for both the local and the global score schemes.To further study the effect of varying the model parameter, we calculated an "error" which quantifies the difference between simulated and observed entropy profiles (see Methods). In Figure ω. We also show results for a simulation performed using the JTT model [ω around the optimum value.Although entropy is commonly used to evaluate sequence conservation in an alignment -38 and tTT model  of evoluω = 0.12, that is in good agreement with the optimum cut-off determined from the acceptance rates, as explained previously. In contrast, for the global-score case the cut-off at the maximum of the similarity score plot is significantly below the optimum ω value previously obtained. This difference would be due to the wrong behaviour of the global-score scheme for small amounts of divergence .Even though it has long been recognized that substitution patterns are site-specific and depend on protein family, it is in general very difficult to estimate site-specific and family-specific substitution matrices due to a lack-of-data problem. As we reported previously, a possible strategy to overcome this obstacle is to obtain site-specific substitution matrices from SCPE simulations . To furtω plots obtained using local-score and global-score SCPE substitution matrices. The global-score likelihood peaks near ω = 0.18 in good agreement with the previous determinations, showing that it reproduces quite well the amino acid substitution patterns found in real sequences. The best ω of the local-score SCPE likelihood  the maximum likelihood peaks near ω = 0.2, in better agreement with Figures ω = 0.15.In Figure  Figures , and 6. ω range studied. This is due to the fact that site-specific amino acid substitution patterns, especially for constrained structural classes i and i+4, are not well described by general models such as JTT.Figure As a further example of the applicability of the SCPE model, we considered other families of the LβH superfamily  are clearly better than JTT for all sites considered. CAT and SATA behave similarly, though the advantage of SCPE over JTT is less marked. For other families, SCPE  is better than JTT for class i, where the more meaningful results are expected, local and global give very similar results for all families except for LACA where global is better than local.When comparing local-score SCPE with global-score SCPE one finds no definite advantage of either one over the other. For sites We presented in full detail the Structurally Constrained Protein Evolution Model (SCPE), developed recently. We improved on our previous model by introducing a new scoring function. Our previous work was based on a "global" score, which measures how a trial sequence differs from the ancestral sequence in its ability to fit a reference structure assumed constant. In contrast, the "local" score measures the perturbation introduced by a given mutation with respect to the previously accepted sequence, rather than the ancestral one.Both schemes, global and local, were compared in their ability to match the substitution patterns of the protein family LPXA. We performed a thorough assessment comparing structure conservation, entropy profiles, amino acid distributions, and substitution matrices. LβH proteins were found to be particularly suited for such a detailed characterization of the sequence pattern, because of the fact that most of their sites belong to one of only six different structural classes. Furthermore, these properties were studied as a function of the single parameter of the model: a cutoff that measures selection pressure against structural divergence. Finally, we applied the model to all other members of the LβH superfamily whose structure is known, extending previous studies performed only on the LPXA family.i of all other families studied, both SCPE models clearly outperform the widely used JTT model, showing the power of the SCPE model to account for substitution patterns conditioned by structural constraints.In general, we found that the local-score SCPE behaves either similarly or better than the global-score scheme, depending on the property considered. Furthermore, for LPXA, and for sites of the structurally constrained class Currently, we are using the SCPE model to investigate several issues important in protein evolution, such as overdispersion of the molecular clock, correlation between the evolution of different sites, and heterotachy. Also, we are testing the applicability of the SCPE model to other protein families, in order to assess its generality. Nevertheless, we should mention that since most protein families do not display the regularity of LβH proteins, it is more difficult to perform a detailed quantification of sequence patterns, which makes such tests at the same time more difficult and less demanding than the LβH superfamily.3- [ASCVTN]-X. The first position of the hexapeptide is called i, and the following i+1, i+2, up to i+5. The sequence forms a left-handed parallel β helix, forming an equilateral triangular prism [i and i+4, for example, point towards the inside of the β helix  is the mean-field energy of position p in the trial (mutated) sequence and Eref(p) is the corresponding value of the reference sequence. The "global score" is calculated using the ancestral sequence as reference. The "local score" is calculated using the sequence accepted in the previous step in the simulation .where N is the length of the protein sequence, Escherichia coli. The coordinates were obtained from the PDB database [The ancestral sequence was the UDP-N-acetylglucosamine acyltransferase (LPXA) from database   and the total number of amino acid mutation trials. We use "calculated", as opposed to "inferred" to designate the acceptance rates obtained in this way.In the SCPE simulations, we also estimated Some of the comparisons performed depend on the amount of divergence. For these cases, we estimated the average divergence of the LPXA family using the program PAML. Maximumλ = 1.10 (ω = 0.15); local-score SCPE with λ = 8.00 (ω = 0.92); global-score SCPE with λ = 7.00 (ω = 0.19); global-score SCPE with λ = 90.00 (ω = 0.95). To compare, we also ran simulations using JTT. For each model, we performed 50 independent runs of lengths Ka = 0.28 and Ka = 1.7 amino acid substitutions per site. For each sequence, structure recognition using THREADER 3 was performed. The ability of models to conserve structure was measured by the percentage of sequences which recognized correctly (Z-score > 2.7) the LβH fold.We evaluated whether sequences produced by evolutionary simulations using SCPE recognize the correct structure using THREADER 3 . We consc = 1,2,...6 site classes. Then, for each class we set up a matrix of counts: for i ≠ j,  is half the number of mutational steps that result in either i → j or j → i amino-acid replacements at site class c, and  is the number of mutational steps for which amino acid i remains constant (i → i replacement). Then, for each class, the matrix of substitution rates, Qc, is obtained using:Site-specific replacement matrices are obtained straightforwardly by "counting" substitutions in SCPE simulations. For the test system considered, sites can be classified into Qc, the probability matrices are obtained usingGiven the rate matrices, Pc = exp(tQc)c is, then, obtained withThe vector of amino acid equilibrium frequencies of class Qc using a pseudocounts procedure similar to that developed by Tatusov [Since there are some substitutions that do not occur during the simulations (very low probabilities), we have found it convenient to re-calculate each  Tatusov  as follo and  are, respectively, the substitution matrix elements and equilibrium frequencies of a reference model. Here we used JTT [α = 0.01. Accordingly, equilibrium frequencies were also corrected usingwhere used JTT  and α = To study the sequence variability profile, we calculated the entropy for each structural class using: is the probability of finding residue i at structural class c.where For SCPE, we used the equilibrium probabilities obtained from the substitution matrices, as described in the previous section. For the reference alignment, we grouped all columns of the same structural class together, counted the number of times each amino acid occurred in each class, and obtained the corresponding amino acid frequencies., and the profile of the observed reference family, , was quantified by the following "error" function:The difference between the entropy profiles obtained from the SCPE models, To assess the similarity between the equilibrium SCPE amino acid distributions and those obtained from the reference alignment, we used the similarity score based on information theory proposed by Yona and Levitt . The scoThe equilibrium SCPE distributions and their corresponding entropies were compared with JTT distributions and entropies. In contrast to SCPE, the equilibrium JTT distribution does not depend on structural class. Therefore, instead of the equilibrium distributions, we chose to use the distributions and entropies from the alignment of sequences obtained from simulations with the JTT model. To this end, we performed 100 independent simulations using the JTT substitution matrix. The simulation length was set to the average number of substitutions obtained for the LPXA family (Ka = 0.28). We aligned the 100 output sequences, grouped all columns of the same structural class together, counted the number of times each amino acid occurred in each class, and obtained the corresponding amino acid frequencies.Qc. The maximum likelihood of a model, Q, given the data, s, for topology, T, is obtained by maximizing the probability L = Pr(s|T, Q).In order to assess the SCPE substitution patterns, we performed Maximum Likelihood (ML) calculations using the site-dependent SCPE substitution matrices, Qc matrices, we calculated the maximum likelihood using PAML. In all cases a gamma distribution was used to take into consideration the rate heterogeneity among sites of the same class. Similarly, we performed ML calculations using the JTT substitution matrix with gamma distribution of rates (JTT+Γ), for each of the six structural classes. The ML values obtained for each class were added together to obtain the total ML, as was done with the SCPE models.For the SCPE model, the reference alignment was partitioned into 6 sub-alignments corresponding to the 6 structural classes. Using these sub-alignments and the corresponding SCPE It has been shown that as long as the tree topology is reasonable, model comparison is robust with respect to variations in topology . In the All the models compared here have the same number of parameters. Therefore, models were compared by comparing ML values. One should note, however, that when models with different number of parameters are compared, one should use a statistic that takes explicit account the number of parameters of each model ,43.GP and JE developed the mathematical model. GP implemented the model, run the simulations, performed the analysis and wrote the first draft. JE edited and wrote the revised versions. All authors read and approved the final manuscript.


Some haematological diseases are associated to an increased risk of thromboembolic events. We report a case of paroxysmal nocturnal haemoglobinuria (PNH) in which a cerebrovascular event represented the first clinical manifestation of disease. PNH is associated to thromboembolic events, generally of venous districts often involving unusual locations such as mesenteric vessels, sagittal veins, inferior vena cava and renal veins.To our knowledge arterial thrombotic episodes are rare and the involvement of arterial cerebral vessels is exceptional. Then, our case points out the importance of investigating about haematological disorders in all patients presenting with a stroke, in which the common predisposing conditions are excluded. Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal disorder of haematopoietic stem cells clinically characterized by acute intravascular haemolytic crisis, in particular nocturnal, often overlapped to chronic haemolysis, and by thrombotic events and bone marrow failure. It is associated with a somatic mutation in the phosphatidylinositol glycan complementation class A (PIG-A) gene, mapped to the X chromosome; the subsequent deficiency of glycosylphosphatidylinositol (GPI) and of GPI-anchored molecules, as the decay accelerating factor (DAF or CD55) and the membrane inhibitor of reactive lysis (MIRL or CD59), causes an increased susceptibility to complement-mediated lysis of erythrocytes, leukocytes and platelets .The association between PNH and thromboembolic accidents, generally manifesting as thrombotic events of venous vessels sometimes complicated by pulmonary embolism, is well established. Arterial thrombotic episodes, particularly of cerebral vessels are enough rare .We report a case of PNH presenting with thromboembolic events, both venous  and arterial (stroke).A 56-year-old woman, with history of peptic ulcer and family history for cerebrovascular disease was referred to our Division of Internal Medicine with asthenia and generalized discomfort. She reported a cerebrovascular accident manifesting as a right brachial and crural hyposthenia ten month ago, almost completely receded at observation time; she also referred recurrent episodes of proximal deep venous thrombosis (DVT) of lower limbs in the last seven months.In order to identify any hypercoagulable state (i.e. inherited or acquired thrombophilia), in view of her personal and familiar history, we tested prothrombin time, as INR, activated partial thromboplastin time, as ratio, fibrinogen, protein C and S, antithrombin III, activated protein C resistance, anti-cardiolipin antibodies IgG and IgM, lupus anticoagulant, plasminogen activator inhibitor type 1, d-dimer, gene polimorphism of clotting factor II and V, gene polimorphism C9774T and G3775A of apolipoprotein B and gene polimorphism C3932T and C4070T of apolipoprotein E resulted all in normal range; while gene polimorphism of tetrahydrofolate reductase and angiotensin converting enzyme revealed heterozigosity for both. Subsequently, homocysteinemia test revealed mild hyperhomocysteinemia. All thrombophilic tests are summarised in table A magnetic resonance imaging scan showed little and multiple ischemic lesions in particular in left cerebral peduncle fig , semiova3, hemoglobin 7.9 g/dl, hematocrit 25%, mean corpuscular volume 99,6 fl, mean corpuscular hemoglobin 32 pg, mean corpuscular hemoglobin concentration 32 gr/dl, white blood cells 4,040/mm3, platelets 93.000/mm3, reticulocytes 5,4%, serum iron 76 μg/dl, erytro-sedimentation rate 1° hour 40 mm, lactate dehydrogenase 944 UI/l, total bilirubin 0,72 mg/dl, indirect bilirubin 0,36 mg/dl, and presence of hemoglobinuria. Coombs' test, cold agglutinins, antinuclear antibodies, anti-extractable nuclear antigens antibodies, anti-mithocondrial antibodies, anti-smooth muscle antibodies were negative. Laboratory data and their range are summarised in table Other available data showed: red blood cells 2.470.000/mmAn abdominal ultrasonography excluded a hypersplenism and/or Kasabath-Merritt syndrome.A peripheral blood smear did not show any finding suggestive for haematological disorders. A bone marrow biopsy showed a slight hyperplasia of erythrocytic bone marrow cell line.These laboratory and morphological findings suggested a non-immune haemolytic anemia. In particular, due to the exclusion of other non-immune haemolityc disorders by means of age and clinical history together with the presence of hemoglobinuria and pancytopenia, we hypothesized paroxysmal nocturnal hemoglobinuria. This diagnosis was confirmed by an immunophenotypic profile of peripheral blood cells, showing a 15% of deficient CD59 erythrocytes, and by the presence of hemosiderinuria. Haematological findings are summarised in table During her hospitalization two haemotrasfusions were necessary in occasion of two concurrent haemolytic crises. Following dismission, in order to prevent further thromboembolic events, the patient began oral anticoagulation therapy with warfarin according with INR value in range of 2–2,5. Moreover she was treated with B12 vitamin and folate supplementation.The association between haematological diseases and thromboembolic events is well established. In particular high thrombotic risk is recognized in patients with essential thrombocythemia, polycythemia vera, PNH and drepanocytosis . PNH is The mechanism whereby PNH causes an hypercoagulable state is not clear. PNH platelets lack the GPI-linked proteins CD55 and CD59, and respond to the deposition of terminal complement components by vesiculations of portions of their plasma membrane, resulting an increased procoagulant property. PNH cells also lack the receptor of the GPI-linked urokinase plasminogen activator, which may result in impaired fibrinolysis . Also anIn our case, thrombotic events represented the clinical onset of PNH and involved both venous (DVT) and arterial (stroke) vessels. Neurological manifestations in PNH patients are generally due to cerebral venous thrombosis ,8, even Also haemotological findings agree with PNH diagnosis because of the association of thrombosis, anemia and thrombocytopenia. We excluded further causes of non-immune haemolityc anemia (i.e. spherocytosis, enzymatic disorders, microangiopathic anemia) and thrombocytopenia .In conclusion, PNH is associated to thromboembolic events, especially in the venous district and should be considered as a possible cause of an hypercoagulable state, in particular when unusual vascular locations are involved. Our case indicates the possibility of arterial thrombotic episodes in a patient with PNH and suggests a thorough evaluation of any haematological disorders in patients presenting with stroke or myocardial infarction, especially in the absence of atherosclerosis risk factors and/or a thrombophilic state.


Previous studies have shown that atherosclerosis of the descending aorta detected by transesophageal echocardiography (TEE) is a good marker of coexisting coronary artery disease. The aim of our study was to evaluate whether the presence of atherosclerosis on the descending aorta during TEE has any prognostic impact in predicting cardiovascular events.The study group consisted of 238 consecutive in-hospital patients referred for TEE testing  with a follow up of 24 months. The atherosclerotic lesions of the descending aorta were scored from 0 (no atherosclerosis) to 3 (plaque >5 mm and/or "complex" plaque with ulcerated or mobile parts).Atherosclerosis was observed in 102 patients, (grade 3 in 16, and grade 2 in 86 patients) whereas 136 patients only had an intimal thickening or normal intimal surface. There were 57 cardiovascular events in the follow-up period. The number of events was higher in the 102 patients with (n = 34) than in the 136 patients without atherosclerosis (n = 23, p < 0.01). The frequency of events was in close correlation with the severity of the atherosclerosis of the descending aorta. Fifty percent of the patients with grade 3 experienced cardiovascular events. Excluding patients with subsequent revascularization, the multivariate analysis only left ventricular function with EF < 40% (HR 3.0, CI 1.3–7.1) and TEE atherosclerotic plaque >=2 (HR 2.4, CI 1.0–5.5) predicted hard cardiovascular events.Atherosclerosis of the descending aorta observed during transesophageal echocardiography is a useful predictor of cardiovascular events. During the past decades many methods and factors have been proposed as good prognostic tools or markers for cardiovascular risk stratification. However, even with the most sophisticated stress testing procedures the prediction capability still remains imperfect . Previound Department of Medicine and Cardiology Center, University of Sciences, Szeged, Hungary. The patients underwent TEE examination for the following reasons: TIA or suspected cerebral embolism (n = 100), coronary flow reserve evaluation (n = 71), evaluation of the native or artificial mitral valve (n = 23), suspected endocarditis (n = 15), evaluation of the aortic valve (n = 13), suspected aortic dissection (n = 6), atrial septal defect or patent foramen ovale (n = 6), others (n = 4). Thirty patients had suffered a previous myocardial infarction. The patients were followed-up to a period of at least 2 years, median 31 ± 9 months.During the year 1998, 238 consecutive patients  were studied with transesophageal echocardiography at the 2All patients had a transthoracic echocardiographic examination to assess the global and regional left ventricular function. The ejection fraction was calculated using the area-length single plane method . Left veThe TEE examination was carried out according to recommendations of the Mayo clinic procedure . Two-dimGrade 0 – no sign of atherosclerosisGrade 1 – intimal thickeningGrade 2 – plaque < 5 mmGrade 3 – plaque > 5 mm and/or "complex" plaque with ulcerated or mobile parts.Significant atherosclerosis was considered in case of grade 2 or 3 fig , 2, 3, 4Follow-up data were obtained from at least one of four sources: 1) review of the patient's hospital record; 2) personal communication with the patient's physician and review of the patient's chart; 3) a telephone interview with the patient conducted by trained personnel; or 4) a staff physician visiting the patients at regular intervals in the outpatient clinic. Follow-up data were obtained in all patients. The outcome events were: cardiac related death, fatal stroke, non-fatal myocardial infarction and revascularization either by percutaneous transluminal coronary angioplasty (PTCA) or by coronary artery by-pass grafting (CABG). Myocardial infarction was documented by a consistent history, EKG changes and cardiac enzyme level elevations and confirmed by hospital chart or hospital discharge letter review. In the next step, patients with revascularization were censored and the remaining patients were considered for the analysis of hard events . All-cause mortality (cardiovascular death plus death of other causes) was also analyzed.Values are expressed as mean ± Standard Deviation. Continuous variables have been compared by the means of Student's t test (two-tailed). Statistical analysis of discrete variables has been performed with chi-square test; a Fisher's exact test has been used when appropriate. The individual effect of certain variables on infarction-free survival has been evaluated with the use of the Cox proportional hazard model with univariate and multivariate analysis and with the Kaplan-Meier method. The patients were stratified into two subgroups: patients with and without cardiac events. The examined variables were age, sex, risk factors , previous myocardial infarction, ejection fraction, and significant atherosclerosis of the descending aorta. A p value <0.05 was considered statistically significant.The examination was successful and complete in all patients, without any side effect. Significant atherosclerosis of the descending aorta was observed in 102 patients, (grade 3 in 16, and grade 2 in 86 patients) whereas 136 patients had only mild intimal thickening (n = 46) or normal endocardial surface (n = 90).There were 57 events in the follow-up period: cardiac related death: n = 14, fatal stroke: n = 4, non-fatal myocardial infarction: n = 5 and coronary artery revascularization: n = 34. Ten patients died of non-cardiovascular causes and the cause of the death of 2 patients was undetermined.The number of events was significantly higher in the 102 patients with (n = 34) as in patients the 136 patients without significant atherosclerosis (n = 23, p < 0.01). The results of univariate and multivariate analysis are shown in table The frequency of events was in close correlation with the severity of the atherosclerosis of the descending aorta fig . Fifty pPatients with early (<3 months, n = 29) or late (>3 months, n = 5) revascularization have been censored.Nine events have occurred in the 122 patients with TEE score ≤1 and 14 in the 82 patients with TEE score ≥2 (7% vs 17%, p < 0.05) fig . The resAtherosclerosis of the descending aorta observed during transesophageal echocardiography is a useful predictor of future cardiovascular events.Our data are in keeping up with previous findings, showing that patients with atherosclerosis of the thoracic aorta have higher probability of coexisting coronary artery disease -8. In thOne of the most important first steps in stratifying risk among patients with proven or suspected coronary artery disease is the identification of patients at high risk for coronary or vascular events during the course of the next few months or years. To date, left ventricular dysfunction, the number of diseased vessels, and the severity of myocardial ischemia have emerged as important determinants of survival . Our datThis study has several limitations. The study population was highly heterogeneous, reflecting the garden variety of patients referred to the echo lab for transesophageal testing: patients with known or suspected coronary artery disease or cerebrovascular disease coexist with patients with congenital or acquired valvular disease. Therefore, our findings cannot be directly translated into the general population, and further prospective studies are needed at this point to evaluate the prognostic value of transesophageally detected aortic pathology in more sharply defined clinical subsets.The morphological characterization of atherosclerotic plaque has not been performed in our study, neither in terms of plaque content  nor of pGrade I. atherosclerosis of the descending aorta. Intimal thickening.Click here for fileSmall plaque on the descending aorta, corresponding to Grade 2. atherosclerosis.Click here for fileGrade 3. atherosclerosis, with large, multiple plaques.Click here for fileGrade 3. atherosclerosis, plaque with mobile parts.Click here for file


An Outbreak Control Team was formed resulting in extensive epidemiological, microbiological and environmental investigations.On 30th July and a standard case definition was adopted. Using a pre-designed proforma information was gathered from both sick and well subjects. Food specific attack rates were calculated and two-tailed Fisher's exact test was used to test the difference between type of food consumed and the health status. Using a retrospective cohort design univariate Relative Risks and 95% Confidence Intervals were calculated for specific food items.Attempts were made to contact everybody who ate food from the restaurant on 27S Enteritidis phage type 34a and one for S Enteritidis phage type 4.Data was gathered on 52 people of whom 38 developed gastrointestinal symptoms; 16 male and 22 female. The mean age was 27 years. The mean incubation period was 30 hours with a range of 6 to 90 hours. Food attack rates were significantly higher for egg, special and chicken fried rice. Relative risk and the Confidence interval for these food items were 1.97 (1.11–3.48), 1.56 (1.23–1.97) and 1.48 (1.20–1.83) respectively. Interviews with the chef revealed that many eggs were used in the preparation of egg-fried rice, which was left at room temperature for seven hours and was used in the preparation of the other two rice dishes. Of the 31 submitted stool specimens 28 tested positive for In the absence of left over food available for microbiological examination, epidemiological investigation strongly suggested the eggs used in the preparation of the egg-fried rice as the vehicle for this outbreak. This investigation highlights the importance of safe practices in cooking and handling of eggs in restaurants. S Enteritidis, S Typhimurium and S Virchow [Infection due to Salmonella is a major public health problem in England and Wales with reports of over 14,400 infections due to Salmonella in the year 2003 [Salmonella enterica serovar Enteritidis phage types 4, 21 and 6 have been reported in previous outbreaks [S Enteritidis PT 34a infection reported in the literature from United Kingdom. In this report, we present the results of an epidemiological investigation of an outbreak due to this rare phage type associated with a Chinese restaurant in Suffolk, United Kingdom.Although utbreaks , phage tth July 2002, the Suffolk Communicable Disease Control team (SCDC) was informed by the consultant microbiologist that S. Enteritidis had been isolated from stool samples of five patients. All had recently eaten a meal in a local Chinese restaurant. Further enquires revealed that there were more patients with similar food history and gastrointestinal symptoms. An Outbreak Control Team was convened on 31st July and it was decided that a full investigation should be carried out to identify the extent of the outbreak, the probable vehicle of infection and to advise on the appropriate control measures.On 30th July 2002. The information collected included name, address, sex, their symptoms and date of onset. The restaurant provided the list of food items that were served/sold on the day in question. This menu extended to 40 food items. This list was shown to the restaurant patrons and they were asked to state the food items they had eaten. A variety of ways was used to identify further cases including the technique of snowball sampling. This involved asking the patrons whether they were aware of any others who had similar symptoms and had eaten in the restaurant. General Practitioners providing primary care in the area were contacted and were requested to check for patients with gastrointestinal symptoms. The presenting symptoms of the patrons were diarrhoea, headache, abdominal pain and fever.The environmental health department (EHD) staff initially gathered information from people who had become ill on a standard data collection form. In the initial stages of the investigation, it became apparent that all those who became ill had eaten or had bought a take away at the restaurant on 27th July whether they became ill or not. The restaurant provided table-booking details. The following case definition was adopted for the outbreak. "Symptoms of acute gastroenteritis including one of the following: diarrhoea, vomiting or abdominal pain up to 96 hrs after having had a meal from the said restaurant including takeaway between 22 and 30 July 2002 and/or individuals who have positive stool sample for S. Enteritidis up to 96 hrs after having a meal from the restaurant including a takeaway between 22 and 30 July 2002".The next step involved interviewing all those who had eaten/bought food on 27th July. Eligibility for membership of the cohort was defined as a person having the opportunity to eat any of the food items available on the day.An analytical investigation was carried out using a retrospective cohort design. Efforts were made to identify anyone who ate or bought food at the restaurant on 27Data was entered in to Statistical Package of Social Sciences version 10 . Food spth of July. Environmental sampling was not carried out as this was considered to be of limited value. There was no food left over from 27th July, but three food samples were taken on 30th July and sent for analysis to the food laboratory at Chelmsford Public Health Laboratory. Stool specimens were sent to Ipswich Hospital microbiology laboratory and were cultured for the presence of Salmonella sp. Isolates of Salmonella were forwarded to the Laboratory of Enteric Pathogens at Central Public Health Laboratory, Colindale for phage typing. Standard procedures were adopted for phage typing at the laboratory [Stool samples were requested from all who had eaten food from the restaurant on 27boratory .The EHD staff inspected the premises including verifying the procedures for hazard analysis and critical control point (HACCP). Egg storage and preparation of egg items were also investigated during the visit. Efforts were made to trace the egg trail back to the supplier.th July of whom 38 developed symptoms and 14 were free of symptoms. Of the 38 who became ill 16 were male and 22 were female. The mean age was 27 years. The mean incubation period was 30 hours with a range of 6 to 90 hours suggesting a point source outbreak. Two patients received hospital care and there were no deaths. No gastrointestinal illness was reported among the kitchen staff of the restaurant in the weeks before or during the outbreak.Data were gathered from 52 subjects who had eaten food from the restaurant on 27On investigation of food preparation practices at the restaurant, it appeared dishes containing egg were the most likely vehicle for this outbreak. However, this information was not discussed when gathering data from the subjects. Data was gathered in a standardised format from all subjects to avoid any interviewer or recall bias.We had a strong "a priori" hypothesis that people who had eaten egg or food that had come in to contact with egg were at an increased risk even before looking at the data and these were analysed first. During analysis it became apparent that illness was significantly associated with the three food items that contained egg or the egg rice mixture as shown by the food specific attack rates Table  and the S. Enteritidis was isolated in 29. Twenty-eight of these 29 isolates were confirmed to be PT 34a and one as PT4. No pathogens were isolated from food samples taken from the restaurant on 30th July.A total of 31 stool specimens were submitted to the laboratory from which th of July the egg rice mixture was left at room temperature for seven hours. The restaurant received eggs from a supplier in London every week and they were not refrigerated. Attempts to trace the egg trail were not successful.During the visit to the restaurant, the EHD staff reported that there was no evidence of hazard analysis and noted many cleaning and maintenance issues. Hand wash facilities were inadequate. The chef explained that after preparing the egg rice mixture, it was left out at room temperature for the rest of the evening, and reheated when ordered. This mixture was used for some of the other fried rice items. It was estimated that on the evening of the 27st July and the EHD staff reassessed the situation on the evening of 1st of August. As they were satisfied with the arrangements, the restaurant was allowed to reopen. The owner and restaurant staff were provided with information on proper cooking methods and the importance of undertaking HACCP.The restaurant closed voluntarily on 31The epidemiological investigation showed that eggs used in the preparation of egg-fried rice, which in turn was used in the preparation of some of the other rice items was the vehicle of infection. Isolation of the unusual phage type 34a strengthened the conclusion that eating from the restaurant was linked to a point source outbreak. In general, by the time investigations are initiated often no food material is available for laboratory analysis and the investigator has to rely on epidemiological evidence. The first step in identifying the source of an outbreak is the calculation of attack rates and the responsible food should have a significantly higher attack rate . In thisStatistical methods have better power while there is an "a priori" hypothesis as shown in the study of summer excess of leukaemia . We had S. Enteritidis outbreaks associated with Chinese food businesses in England [To our knowledge, this is the third report of an outbreak due to phage type 34a and the first of its kind in England published in the literature. In the UK, this phage type has been associated with travel abroad especially to southern Spain  and indi England , Scotlan England  and the  England  although England , egg rolS. Enteritidis has been reported to be low [S. Enteritidis in causing food borne outbreaks is well known as it has the ability to contaminate eggs without causing discernible illness in the birds affected [et al [The proportion of eggs infected with o be low  and hencaffected . Eggs haaffected -22. Hayed [et al  in theirS. Enteritidis phage types appear to be genetically related or clonal. Discussion on stability of phage types of S. Enteritidis can also be found in the literature. Conversion of phage type 4 to 24, phage type 23 to 8 and 4 to 7 have been reported. We could not find reports linking phage type 34a and phage type 4. In a recent public health investigation [S. Enteritidis in raw eggshells, various serotypes of Salmonella were isolated from 23 out of 449 (5.1%) pooled samples labelled as originating from Spain. These sero/phage types included S. Enteritidis PT6a, PT5c, 13a, 14b, 58, PT6d, PT1, PT1c and PT12.We could not find any veterinary data on phage type 34 in British flocks. We searched the literature to determine whether there is any molecular relationship between phage type 34a and phage type 4. Hudson et al  based ontigation  of S. EnA few limitations of this study are to be noted. The origin of the suspected contaminated eggs could not be traced. Although trace back exercises are key in epidemic investigations, often they are not successful due to logistic and practical reasons. In our outbreak one of the suppliers to the restaurant turned out to be a packaging firm. During our investigation, we found that there were problems with the distribution system, which prevented us from pin pointing the origin of the contaminated eggs. A possibility always exists that we missed a few subjects from this investigation and not all could be persuaded to provide a stool sample. There was no single list of all the patrons who ate/purchased food on the evening. However, all efforts were made to contact the patrons and the outbreak caused considerable publicity in the local media. Hence, we are confident that we have included most patrons. Although the precise number of patrons who were not included will never be known we are confident that their number is small and might be in the region of 10 to 15.Palmer  has poinS. Enteritidis PT4 was isolated from one of the subjects. Further investigation revealed that this subject had recently returned from holiday in continental Europe and had suffered mild symptoms before the meal.In response to this and other outbreaks associated with eggs, a Public Health Investigation was launched in October 2002 in the UK to determine the rate of Salmonella contamination in eggs. Tests of nearly 4000 eggs showed that Salmonella was recovered from 5.3% of pooled eggs . The FooInvestigation of this outbreak was greatly facilitated by the close cooperation between local EHD, Communicable Disease Control Team, microbiological laboratory and local health care providers. Although food samples from this point source outbreak were not available for microbiological culture, epidemiological evidence pointed to eggs containing dishes as the most likely source of the outbreak. This outbreak highlights the continuing hazards of raw eggs. It is likely that the use of pasteurised eggs and the adoption of safe food preparation practices would have prevented this outbreak.The first author (PB) served as the expert witness for the prosecution during the court proceedings.PB and TS conceived and designed the study and drafted the manuscript.PB analysed the dataRK oversaw the microbiological investigation.PB, TS, RK and HM all interpreted the results of the analysis and critically reviewed the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Tertiary care hospitals are a potential source for development and spread of bacterial resistance being in the loop to receive outpatients and referrals from community nursing homes and hospitals. The liberal use of third-generation cephalosporins (3GCs) in these hospitals has been associated with the emergence of extended-spectrum beta- lactamases (ESBLs) presenting concerns for bacterial resistance in therapeutics. We studied the 3GC utilization in a tertiary care teaching hospital, in warded patients  prescribed these drugs.Clinical data of patients (≥ 13 years) admitted to the General Hospital, Port of Spain (POSGH) from January to June 2000, and who had received 3GCs based on the Pharmacy records were studied. The Sanford Antibiotic Guide 2000, was used to determine appropriateness of therapy. The agency which procures drugs for the Ministry of Health supplied the cost of drugs.The prevalence rate of use of 3GCs was 9.5 per 1000 admissions and was higher in surgical and gynecological admissions (21/1000) compared with medical and orthopedic (8 /1000) services (p < 0.05). Ceftriaxone was the most frequently used 3GC. Sixty-nine (36%) patients without clinical evidence of infection received 3Gcs and prescribing was based on therapeutic recommendations in 4% of patients. At least 62% of all prescriptions were inappropriate with significant associations for patients from gynaecology (p < 0.003), empirical prescribing (p < 0.48), patients with undetermined infection sites (p < 0.007), and for single drug use compared with multiple antibiotics (p < 0.001). Treatment was twice as costly when prescribing was inappropriateThere is extensive inappropriate 3GC utilization in tertiary care in Trinidad. We recommend hospital laboratories undertake continuous surveillance of antibiotic resistance patterns so that appropriate changes in prescribing guidelines can be developed and implemented. Though guidelines for rational antibiotic use were developed they have not been re-visited or encouraged, suggesting urgent antibiotic review of the hospital formulary and instituting an infection control team. Monitoring antibiotic use with microbiology laboratory support can promote rational drug utilization, cut costs, halt inappropriate 3GC prescribing, and delay the emergence of resistant organisms. An ongoing antibiotic peer audit is suggested. Appropriate use of antibiotics is central to limiting the development and the spread of resistant bacteria in hospitals and communities. Use of broad-spectrum antibiotics, in particular the 3GCs in nosocomial infections have been linked to the emergence of antibiotic resistance and increase in costs . The hosEscherichia coli and Klebsiella spp. An important source of this resistance results from the production of extended-spectrum beta-lactamases (ESBLs) by bacteria. ESBLs are modified beta-lactamase enzymes mainly derived from the ubiquitous TEM1/2 and SHV-1 plasmid-mediated enzymes, which hydrolyse expanded spectrum cephalosporins to varying degrees. Many beta-lactamases result in resistance to 3GCs in Enterobacteriaceae. Genera such as Enterobacter, Citrobacter and Serratia posses chromosomal broad spectrum beta-lactamases which are normally repressed, and when induced result in resistance to 3GCs. Klebsiella and E. coli usually have the SHV- or Tem- type beta-lactamases, and key mutations in these result in true "ESBLs". ESBLs have received attention in the last decade because although penicillins, cephalosporins, or aztreonam appear to be susceptible in vitro, ESBL producing E. coli or Klebsiella spp. may demonstrate clinical resistance to these antibiotics leading to treatment failures. Liberal use of the 3GC antibiotics has resulted in the ESBLs conferring resistance among Enterobacter[K pneumoniae and other Enterobacteriaceae and vancomycin-resistant enteroccocci with cephalosporin use [Enterobacteriaceae has been reported from Barbados [Enterobacteriaceae has recently been observed in tertiary care in Jamaica [The increasing resistance to 3GCs accompanied by an increasing cost burden has raised concerns about the detection, prevalence, and clinical implications of infections with erobacter-6 comproorin use -11. ReceBarbados , and ext Jamaica  and Trin Jamaica . From unIn Trinidad and Tobago, health care is delivered through the public sector, though several private facilities are also available. At the publicly-financed health care institutes patients undergo investigations and receive treatment without cost. There are 2 tertiary general hospitals in Trinidad, the General Hospitals at Port of Spain and San Fernando and several secondary and primary health care facilities where patients can access medical care. The POSGH is the country's major health care institute and receives referrals from all over the country including the sister isle of Tobago. This hospital is a 900-bed institute providing out-patient services and health care for warded patients for over one third of Trinidad and Tobago's population of 1.3 million. Medical students undergoing training at the University of the West Indies, also attend clinical rotations at this hospital.Between January to June 2000, we conducted a cross sectional study of adult in- patients (over 13 years) who had received one or more courses of treatment with one of the 3GCs (cefotaxime, ceftriaxone, ceftazidime) available at the POSGH. The Hospital Pharmacy identified warded patients from the respective services based on the prescriptions dispensed. Patient characteristics, clinical data and laboratory investigations were obtained from the hospital records. While we did consider the course of antibiotic duration in defining appropriateness of therapy, we did not analyse data based on use per patient days because these data were not clearly available from the records. Specific data on the category of service, concomitant disease and drug therapy, organ system with infection, invasive/indwelling devices and the 3GC used were collected using a standardized instrument.An infection was deemed to be present when the physician's diagnosis of infection was a differential diagnosis stated in the chart and/or in a patient who had a fever (>100.4 C) and elevated WBC Count >12,000/cmm.On review all patients with diabetes and HIV had a clinical diagnosis of infection stated in the records.The appropriateness of antibiotic therapy was determined using the criteria described in the Sanford Antibiotic Guide, 2000. Therapy was deemed to be inappropriate based on any of the following parameters: the type of therapy (prophylactic and empiric), combination of antibiotics, the route of administration, the dose, and the duration of therapy. The Sanford guide is widely accepted in Trinidad and the Caribbean as a reference for making appropriate therapeutic recommendations and is also used as a reference manual by medical microbiologists. No other guidelines are currently available in Trinidad. The course of therapy was considered a parameter of appropriate therapy, but a separate analysis based on this parameter was not possible since the information was not always available from the patient records.The direct cost of total antibiotic treatment was computed using the data obtained from the National Insurance Property Development Corporation, which is the agency contracted by the Ministry of Health for procurement and distribution of drugs in the public sector health institutes.2) tests.Data were analysed using EPI Info 6.4 (CDC, Atlanta GA) and categorical variables were compared using the Odds Ratio (95% CI) and the chi-square (XOne hundred and ninety two (192) adult patients admitted to the POSGH were treated with 3GCs during the first six months of 2000, providing a prevalence rate of use of 9.5 patients per 1000 admissions, (192/20146). The rate of use was higher (p < 0.05) in those patients utilizing the surgical and gynecological services (21 per 1000 admissions) compared with those admitted in the medical and orthopedic services (8 per 1000 admissions). One hundred and twenty seven (66%) patients received ceftriaxone, cefotaxime was prescribed for 51 (26.5%) patients, and ceftazdime was administered to 14 (7.5%) patients.The demographic characteristics of patients who received a 3GC and factors related to infection and treatment in the study population are shown in Table There were 107(56%) patients who had fever and a high WBC count with leucocytosis and who were considered to have a clinically suspected infection. Thirty six percent of patients who had no clinical evidence of infection had received treatment with a 3GC. The most common site of suspected infection (20.3%) was the skin and soft tissue followed by the respiratory (10.9 %), gastrointestinal (10.7%), and urinary (9.3%) tracts.Sixty eight percent (131) of patients in this study received empirical antibiotic treatment and in 29.7% of patients these antibiotics were prescribed as prophylactic therapy. Only 4% of patients received 3GCs based on recommended therapeutic regimens. More patients (87%) who were admitted to the medical services received empirical antibiotic treatment compared with patients in the surgical services (68%), (p < 0.05). Prophylactic antibiotic regimens were most frequently prescribed in the orthopedic (50%) and gynecological services (45%) in contrast to patients in surgery (31%) who received 3GCs as prophylactic therapy. However patients in the surgical units (61%) were more likely to be administered two or more antibiotics compared with those receiving medical care in the orthopedic (33%) or gynecological (45%) services (p < 0.01). Single antibiotic therapy was prevalent in the medical, orthopedic, and gynecological services (66%, 55%, and 55% respectively). Seventy-eight percent of those patients who did not display any clinical evidence of infection were treated with one antibiotic. Patients with infections of the skin and soft tissue (75%) and the urinary tract (61%) were more likely to receive treatment with two or more antibiotics compared with patients admitted with respiratory tract infections (48%) (p < 0.05).Pseudomonas spp (7), Acinetobacter spp (3), Enterobacter spp (2), Klebsiella spp (2), Proteus (2) S. aureus (3), and others (8).Biological samples were not sent to the laboratory in as many as 73% of patients to determine a bacteriological etiology of suspected infection. Table . Of the An analysis of factors that were associated with the inappropriate use of 3GCs in the patient sample is shown in Table The average duration of patient stay in the hospital was 14 ± 22 days (range 1 day -150 days). Patients in the orthopedic wards were hospitalized for more days (24.5 ± SD 35.28) compared with the number of days patients spent in the medical (12.45), surgical (13.7) and gynecology (7 days) services. Interestingly, the mean duration of stay of patients in whom antibiotic treatment was appropriate, was not significantly different from the number of days that patients who were treated inappropriately were warded. The overall direct costs from use of these antibiotics in this study was TT$ 117,432 (US$ 19251.00). The cost of treatment with these antibiotics in patients who were inappropriately treated was (TT $79,487.00, US$ 13, 30.00) and was twice as high as the cost of treatment for those patients who were treated appropriately (TT$ 35, 138.00, US$ 5767)The extensive use of third generation cephalosporin antibiotics has caused the emergence of extended spectrum beta-lactamases in Gram-negative bacteria worldwide -6. More In our study, the prevalence rate of use of 3GCs was lower than the reported rate of 43 per 1000 admissions for ceftriaxone in 51 Victorian Hospitals in Australia of which 82% was prescribed as empirical treatment . Our datEnterobacter spp to cefotaxime and ceftriaxone was 53% and 37% respectively in the Intensive Care Unit at this hospital. It is interesting that only 4% of prescriptions were based on therapeutic regimen following culture and sensitivity results in our study, and in 73% of patients with suspected infection bacteriological confirmatory tests were never done and 3GCs were prescribed. We believe high inappropriate use of these antibiotics has contributed to increasing multiple antibiotic resistance and extended spectrum beta-lactamase producing enterobacteriaceae observed in this hospital [Third generation cephalosporins were introduced in the Caribbean between the late eighties and the early nineties and soon after in 1993, the first isolation of resistant Gram-negative bacilli resistant to them was demonstrated in Barbados . In Jamahospital .The cost of inappropriate antibiotic use was twice as much for patients who were treated appropriately and highlights irrational antibiotic consumption at the hospital, prompting protocols for rational antibiotic prescribing and utilization review. The World Health Organization has recommended multifaceted strategies to improve hospital-prescribing practices such as the development of consensus guidelines, educational activities and rapid feedback to prescribers about inappropriate use to reduce overuse of antibiotics in the hospital . RestricThis first audit on 3GC use from the Caribbean demonstrates inappropriate use of these drugs in tertiary care and presents opportunities to develop consensus guidelines for rational use of these drugs in hospitals. We recommend microbiological services in the hospitals undertake continuous surveillance of resistance patterns, to guide in the development of prescribing guidelines. Such guidelines should be widely disseminated and implemented in these institutions, so that prescribers remain informed of rational therapeutics. This study highlights the need to constitute an antibiotic monitoring team comprising a pharmacist, physician, medical microbiologist and infection control nurse to periodically review and evaluate the use and cost of antibiotics at the major tertiary care hospital in Trinidad, to assist clinicians in optimizing clinical care of patients with Gram-negative infections.3GCs Third generation cephaosporinsESBLs Extended spectrum Beta lactamsesPOSGH General Hospital, Port of SpainThe author(s) declare that they have no competing interests.LMPP conceived the study, the design, co-ordinated it and prepared the final manuscript. MP, HR and KT did the data collection. PP assisted with the co-ordination, did the analysis and prepared the draft manuscript. All authors saw the final manuscript and made contributions.The pre-publication history for this paper can be accessed here:


Nicotinic acid is a unique cholesterol modifying agent that exerts favorable effects on all cholesterol parameters. It holds promise as one of the main pharmacological agents to treat mixed dyslipidemia in metabolic syndrome and diabetic patients. The use of nicotinic acid has always been haunted with concerns that it might worsen insulin resistance and complicate diabetes management.We will discuss the interaction between phosphorous metabolism and carbohydrate metabolism and the possibility that worsening of insulin resistance could be related to adrug induced alteration in phosphorous metabolism, and the implications of that in medical management of diabetes and metabolic syndrome patients with mixed dyslipidemia. Nicotinic acid functions in the body after conversion to nicotinamide adenine dinucleotide (NAD) in the NAD coenzyme system. Niacin reduces total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), apolipoprotein B-100 (Apo B) and Lipoprotein (a) (Lp (a)). Niacin increases high-density lipoprotein cholesterol (HDL-C), apolipoprotein A-I (Apo A-I), lipoprotein A-I, and HDL2: HDL3 ratio. The severity and type of underlying lipid abnormality determines the extent of response to niacin treatment [There is evidence suggesting that niacin reduces the risk of a coronary artery or cerebrovascular disease in the setting of secondary prevention, and when combined with a bile acid-sequestrant, promotes regression and decreases progression of atherosclerotic cardiovascular disease ,3.Niacin is known to cause transient, small but statistically significant dose-related reductions in phosphorous levels. These effects were studied for extended release niacin in placebo-controlled trials, mean percentage changes from baseline (± standard error) in phosphorous were -4.0 ± 1.3) for the 500 mg once daily dose, (-7.2 ± 1.1) for the 1000 mg once daily dose, (-9.1 ± 1.1) for the 1500 mg once daily dose, and (-12.6 ± 1.6) for the 2000 mg once daily dose [.0 ± 1.3 It has been long known that niacin induces impaired fasting glucose. I was also suggested that this effect is most likely transient. The mechanism behind this phenomenon has not been fully elucidated .Multiple studies were conducted to study effects of nicotinic acid on exacerbating insulin resistance. One retrospective study suggested that the use of moderate doses of extended release nicotinic acid (average, 1000 mg/d) in reasonably controlled diabetics was associated with improved glycemic control with HbA1c levels decreased by 0.5% ± 0.3% due to aggressive hypoglycemic therapy; as most of these patients had their insulin or oral diabetes regimen intensified . AnotherThe (ADMIT) trial, was one of the first studies to demonstrate the safety of nicotinic acid in patients with diabetes. Diabetic patients  were randomized to treatment with either placebo or niacin (2500 mg/d) for 18 weeks. Patients randomized to niacin therapy had modest increases in fasting glucose (+8 mg/dL). There was no significant change in HbA1c levels or in diabetes treatment regimen in the niacin-treated diabetic patients group .It appears from the clinical data presented above, that nicotinic acid at higher doses could results in alterations in glycemic control of patients with an already abnormal glucose metabolism.Phosphate is primarily an intracellular anion. Insulin is implicated in the transport of glucose and phosphate from the intra-cellular to the extra-cellular space. Studies have suggested a link between decreased serum phosphorous and disturbed carbohydrate metabolism, eventually leading to hyperglycemia ,11. ChroIt seems intuitive to suspect that the transient decrease in plasma phosphorous level might explain the transient disturbance in carbohydrate metabolism and hyperglycemia induced by nicotinic acid. Also, studies suggest the existence of a dose response relationship in both instances.One way to test this hypothesis will be to administer nicotinic acid to both healthy subjects, and to those with impaired fasting glucose or subjects with diabetes, then measure simultaneously at different doses serum phosphorous, and correlate the changes to changes in serum glucose and serum insulin either individually or combined as a measure of insulin resistance.It will be of interest to study if such a relationship exists in patients with the metabolic syndrome diagnosis, since literature suggests the existence of an abnormal phosphorous metabolism in these patients ,12.This hypothesis has several important insights and implications:1. If valid, it suggests the possibility of a simple clinical way to prevent nicotinic acid induced worsening of glycemic control by dietary phosphorous supplementation, eliminating the need to intensify expensive diabetic regimens in diabetic patients that need nicotinic acid treatment.2. If valid, it will aid in lessening the theoretical anxiety of the possibility of precipitating diabetes in glucose intolerant or metabolic syndrome patients in need of nicotinic acid treatment for the management of dyslipidemia.3. Metabolic syndrome and diabetics stand to benefit greatly from the new evidence implicating the importance of increasing HDL-cholesterol in cardiovascular event reduction.4. This hypothesis offers new insights into the mechanism of development of glucose intolerance.


One of the most remarkable capabilities of the adult brain is its ability to learn and continuously adapt to an ever-changing environment. While many studies have documented how learning improves the perception and identification of visual stimuli, relatively little is known about how it modifies the underlying neural mechanisms. We trained monkeys to identify natural images that were degraded by interpolation with visual noise. We found that learning led to an improvement in monkeys' ability to identify these indeterminate visual stimuli. We link this behavioral improvement to a learning-dependent increase in the amount of information communicated by V4 neurons. This increase was mediated by a specific enhancement in neural activity. Our results reveal a mechanism by which learning increases the amount of information that V4 neurons are able to extract from the visual environment. This suggests that V4 plays a key role in resolving indeterminate visual inputs by coordinated interaction between bottom-up and top-down processing streams. In monkeys trained to identify natural images embedded in noise, changes are seen in the information about learned stimuli signaled by V4 neurons It is well established that learning can have a strong impact on neural responses to visual stimuli in high-level association cortices such as inferior temporal (IT) or prefrontal (PF) cortex, where the activity of single neurons reflects learning in pair association, object identification, or categorization tasks . In thesRecent evidence suggests that neurons in early visual sensory areas can also modify their response properties with learning. In particular, several studies have revealed learning-related changes in primary visual cortex (V1) , althougLearning thus appears to affect both low and high level areas of the ventral visual stream. The results obtained by studies in these two areas are, however, difficult to compare directly, owing to substantial differences in experimental design. In studies of IT or PF cortex, studies typically employ ‘complex' visual stimuli such as Fourier descriptors , computeFor a comprehensive account of how learning affects visual processing, the same stimuli and experimental procedure must be used to study different levels of the visual processing hierarchy. What kind of stimuli might be suitable to study visual areas as different as early sensory visual and PF cortex? We decided to use natural images for several reasons: The primate visual system evolved in the natural environment under conditions of ‘natural' stimulation; much is known about their statistical properties and they can therefore be well-controlled; they contain structure at all spatial scales and thus can be expected to activate a large fraction of visually responsive neurons. We avoid subjectively biasing our sample of recorded neurons by always recording from the first neurons whose waveforms we are able to reliably isolate. This ensures that our population of recorded neurons represents an unbiased sample in each brain region under study, and this in turn allows us to compare data obtained from different brain regions. We obtain a sensitive measure of behavioral performance and associated neural activity by employing a stimulus degradation procedure that makes stimuli harder to discriminate by adding various amounts of noise see A. With dHere our aim is to use similar stimuli and behavioral procedures to characterize how learning modifies neural activity in extrastriate visual cortical area V4. Area V4 was chosen because it is considered to be a sensory visual area at an intermediary processing stage in the ventral stream and because it is directly connected to parts of the PF cortex . Our tast-test, p < 0.01). For undegraded stimuli at 100% coherence, the monkeys' performance was near ceiling for both novel and familiar stimuli . Learning-dependent performance improvements for degraded stimuli were highly consistent across stimuli and monkeys. There were in fact no significant differences in the monkeys' performance to each of the familiar stimuli across sessions at all coherence levels (one-way ANOVAs, p > 0.1), and this was also true for novel stimuli. In addition, performance for novel and familiar stimuli did not differ significantly between the two monkeys at any coherence level (t-tests, p > 0.1). Note that the monkeys' excellent perfor-mance with undegraded novel objects reflects the fact that they have acquired the rule of the DMS task and are thus able to perform it near ceiling with novel stimuli. The timecourse of this learning-dependent difference in performance is shown in t-test, p = 0.43). Furthermore, the learning-dependent difference in performance appeared to asymptote after around ten sessions. In summary, learning led to robust improvements in the monkeys' ability to identify degraded natural images while the monkeys performed near ceiling for novel and familiar undegraded images.We found that learning resulted in significant and robust improvements in monkeys' ability to identify degraded stimuli. Behavioral performance varied systematically with coherence A. Monkeynov〉 = 36.7 ± 2.8 Hz) and familiar stimuli (〈FRfam〉 = 34.2 ± 2.7 Hz) was similar , similar fractions preferred familiar or novel stimuli . We thus found that learning did not lead to a change in the average activity of V4 neurons for undegraded stimuli. Next, we examined whether learning resulted in any change in the amount of stimulus-specific information that V4 neurons communicated. To do this, we computed the mutual information between the set of four familiar or novel stimuli and the associated neural responses (nov〉 = 0.48 bits and for familiar stimuli 〈Ifam〉 = 0.45 bits (t-test, p = 0.16). We selected 25% of the population (21 out of 83 neurons), which communicated most information about novel or familiar objects . Thus, for natural images (undegraded stimuli) we saw no significant learning-dependent difference in performance and also no learning-dependent changes in the average activity or in the amount of stimulus-specific information communicated by V4 neurons.We now examine whether there were any learning-depen-dent changes in the activity of V4 neurons. Results described in this report are based on a population of 83 V4 neurons. We first asked whether there was any general difference in mean activity elicited by novel and familiar undegraded stimuli. We found that the response of V4 neurons to novel . For the 25% most informative neurons (white circles in nov〉 = 0.47 bits to 〈Ifam〉 = 0.67 bits (t-test, p < 0.001), corresponding to a 40% increase in information with learning. We further characterized this effect by examining how degradation affected the amount of information separately for novel and familiar stimuli. For both novel (100) than degraded (Idegrad) stimuli (paired t-tests, p < 0.001), reflecting the fact that behavioral perfor-mance was better for undegraded than degraded stimuli (see 100 − Idegrad) for familiar and novel stimuli shown in the insets (t-test, p < 0.001), and learning was associated with a rightward shift in this distribution (〈ΔIfam〉 = 0.06, 〈ΔInov〉 = 0.13). Interestingly, the kurtosis or skewness of the ΔI distribution changed by an order of magnitude from 0.13 for novel stimuli to 5.5 for familiar stimuli, similar to experience-dependent effects that have been observed on hippocampal place cell activity (2 test, p < 0.001). Taken together, learning accordingly resulted in an increase in the amount of information communicated by V4 neurons about degraded stimuli and many neurons actually communicated more information about degraded than undegraded familiar stimuli.At intermediate coherence levels, the monkeys' ability to correctly identify degraded stimuli was improved by learning, and we asked whether this behavioral improvement was associated with any changes in the activity of V4 neurons. We found that V4 neurons indeed communicated significantly more information about degraded familiar than about degraded novel stimuli B. Considth novel A and famth novel B stimulimuli see A. The ΔIe insets A and 4B,activity . As a cot-tests, p < 0.01). For this neuron . By contrast, activity to novel stimuli systematically decreased with degradation and was significantly below activity to undegraded stimuli at coherence levels of 35% and 45% (paired t-tests, p < 0.05). As expected, V4 neurons generally showed greater activity to novel and familiar stimuli than to pure noise at 0% coherence (paired t-tests, p < 0.05). As detailed in t-test, p < 0.05). Taken together, learning resulted in an increase in information communicated by V4 neurons about degraded or indeterminate stimuli. This increase in information was mediated by neurons that showed an enhancement in neural activity to degraded compared to undegraded familiar stimuli.How did single V4 neurons mediate this learning-depen-dent increase in information about degraded stimuli? The activity of an example neuron is shown in 2  for novel stimuli to 1.47 ± 0.16 dva2 for familiar stimuli (unpaired t-test, p < 0.0001). There were also significant learning-dependent increases in the high-probability FPM areas at 45% and 100% coherence . This learning-dependent increase in the high probability FPM regions and their overlap was highly consistent across sessions and monkeys, and we observed it during all six sessions in both monkeys. Note that the lower FPM values for novel stimuli indicate that eye position was less focused and therefore more distributed before learning, whereas for familiar stimuli robust regions of focused eye position developed.We performed additional behavioral experiments to assess whether learning led to any changes in fixational eye movements, because such changes might shed light on what mediates monkeys' behavioral advantages for familiar degraded stimuli. In these studies, we allowed the monkeys to freely view sample stimuli during task performance and then estimated a fixation probability map (FPM) for each familiar and novel stimulus presented at 45% and 100% coherence  levels with decreasing stimulus coherence in area V4 of anesthetized monkeys using novel stimuli  . After Our findings in V4 are in stark contrast to data obtained in the PF cortex using similar task and stimuli . In the The task dependence of learning effects in V1  has beenSeveral computational models have investigated how brain regions might interact during stimulus identification. A key feature of such models is the interaction between bottom-up and top-down processing . ConsideMacaca mulatta) participated in these experiments. All studies were approved by local authorities and were in full compliance with applicable guidelines (EUVD 86/609/EEC) for the care and use of laboratory animals. Stimuli were 10° × 10° in size, 24-bit color depth, and presented at the center of gaze on a γ-corrected 21-inch monitor with linear display characteristics placed at a distance of 97 cm from the monkeys. Stimuli were generated using Fourier techniques that have been described in detail elsewhere . After a delay of 1000 ms, a probe stimulus was presented for 600 ms, which could be any one of the eight undegraded images (100% coherence). The monkeys were required to release the lever if the probe matched the sample . In case of a nonmatch, a second brief delay (200 ms) followed the probe, and this delay was always terminated by the presentation of the correct matching stimulus, ensuring that monkeys had to make a behavioral response on every trial. The monkeys were rewarded with apple juice for making correct responses and were rewarded randomly at 0% coherence where the sample contained no task-relevant information. During each session, the monkeys performed the task with a set of four familiar stimuli, with which they had many weeks of practice, as well as with a set of four novel stimuli that they had never seen before. Matches occurred on 50% of trials; the other 50% were non-matches selected randomly from the remaining stimuli.Owing to the normalization procedure, familiar and novel stimuli did not differ in terms of low-level characteristics of spatial frequency content and image intensity. Familiar stimuli from four categories were used (faces, flowers, birds, and landscapes), and one of the four novel stimuli also came from each of these categories. Fixation was monitored with a scleral search coil and sampled at 200Hz (CNC Engineering, Enfield, Connecticut, United States), and the monkeys were required to maintain fixation within a ±1.25° window at all times during the trial. The monkeys completed at least ten trials per condition during each session.t-test, p = 0.87), but significantly lower during recording sessions for familiar stimuli (t-test, p < 0.01), likely owing to nonspecific factors such as additional wait periods during these sessions.Recordings were made from V4 using standard electrophysiological techniques. We employed a grid system (CRIST, Damascus, Maryland, United States) with eight tungsten microelectrodes (FHC Inc., Bowdoinham, Maine, United States). Preoperative magnetic resonance imaging (MRI) was used to identify the stereotaxic coordinates of V4, which was then covered by a recording chamber. To ensure an unbiased estimate of neural activity, we made no attempt to select neurons based on task selectivity. Instead, we advanced each electrode until the activity of one or more neurons was well isolated and then began collecting data. Comparison of the monkeys' performance during the last six training sessions to performance during recording sessions revealed that performance was unchanged for novel objects . The thresholded FPMs shown in To determine whether there were any learning-related changes in the monkeys' fixational eye movements, we performed separate behavioral experiments in which we allowed the monkeys to freely view the sample stimulus for a period of about 2 s. As before, we presented four familiar and four novel stimuli during each session, but we only used two coherence levels of 100% and 45% to allow us to assess whether learning led to changes both for degraded and undegraded stimuli. Monkeys performed around 20 trials for each stimulus at each degradation levels during each session, and we report here the results from a total of six sessions. We identified periods of fixation during free-viewing as periods as periods of at least 100 ms duration during which eye position did not change by more than 0.3°. We then marked off a region of 0.3° × 0.3° around this position and superimposed these regions for all fixations during all relevant trials. By normalizing the volume under this function to a value of 1, we created an FPM for each stimulus. We then applied a single threshold to the FPM for all stimuli and degradation levels. The threshold η was chosen to be an order of magnitude greater than the FPM value corresponding to randomly distributed eye position, i.e., to a value of η = 10 × 1/256t-test evaluated at p < 0.05. Mean firing rates, reported in Neural activity was assessed during a fixed period of 310 ms duration, beginning 50 ms after the onset of the visual stimulus to take response latency into account. Such a period roughly corresponds to the time between saccades during natural viewing conditions. Out of a total population of 116 neurons, 83 task-related neurons were identified as showing significant differences in activity between any of the eight stimuli at any coherence level using a Bonferroni-corrected s) and the neural responses (r):To assess whether learning had any systematic effect on the amount of stimulus-specific information communicated by V4 neurons, we quantified how much information was contained in the pattern of neural firing rates about novel and familiar stimuli separately. This quantity is given by the mutual information between the set of four familiar or novel stimuli and the set of associated firing rates . We thusP(s) is the probability of showing stimulus s, P(r|s) is the probability of observing a response r when stimulus s is presented, and P(r) is the probability of observing response r.where Because calculation of information requires many trials, we computed information for two conditions: degraded and undegraded stimuli. For degraded stimuli, we pooled the coherence levels from 35% to 65%. For undegraded stimuli, we estimated the mutual information for 100% coherence stimuli during the sample period as well as during the probe period on nonmatch trials (to exclude possible movement-related activity). We report here estimates during the probe period because they are based more trials, but results were similar for the sample period. This ensured that information measures for degraded and undegraded stimuli were based in a similar number of trials.fam,100, Ifam,degrad) and novel (Inov,100, Inov,degrad) stimuli. Note that although across all sessions we employed many more novel than familiar stimuli, each individual neuron from which we recorded during a given session ‘saw' exactly the same number of four familiar and four novel stimuli. We identified highly selective neurons in each population by selecting the 25% neurons that communicated most stimulus information about either novel or familiar stimuli ; i.e., we chose the top 25% of the distribution max(Ifam,Inov). We did this because, owing to our unbiased procedure, our sample contains neurons that did not communicate large amounts of information, and we thus wanted to establish that our conclusions also applied to the neurons that communicated most information. These neurons are shown as white filled circles in n = 62) are shown as gray filled circles. There was significant overlap (13/21, 62%) between the populations of informative neurons for degraded and undegraded stimuli (χ2 test, p < 0.05), indicating that the majority of neurons that were informative for undegraded stimuli were also informative for degraded stimuli. There were no significant differences between informative neurons and the entire population in terms of mean firing rate. Unless otherwise noted, we used paired t-tests to compare information measures obtained for novel and familiar stimuli.For each neuron we estimated four different information values, describing how much stimulus-specific information was contained in its firing rate distributions about undegraded, as well as degraded, familiar (I