Antibiotic surveillance initiatives are limited in resource-constrained settings. In the present study, a quantitative comparison of antibiotic use rates for suspected infections in 2 hospitals in India was performed using the “focus of infection” approach to identify targets for quality improvement in antibiotic prescription patterns in hospitalized patients.This observational study was carried out in one teaching and one nonteaching hospital. All the patients with suspected bacterial etiology were included. Data on the prescribed antibiotics and the focus of infection were prospectively collected using a structured questionnaire. Each diagnosis was further reviewed and confirmed by an independent consultant. The prescribed antibiotics were coded according to the World Health Organization Anatomic Therapeutic Classification (ATC) index with the defined daily dose (DDD) methodology. Focus-specific DDDs were calculated per hundred patient days (DDD/HPD).A total of 6026 patients were included from 72 participating physicians out of available 75 physicians. Overall antibiotic prescribing was higher by 5 percentage points in the teaching hospital (95%) than in the nonteaching hospital (90%). Quinolones (ciprofloxacin constituting 86% of DDD/HPD) were the highest prescribed class in the teaching hospital, and third-generation cephalosporins , in the nonteaching hospital. The targets identified for improvement were the following: longer than recommended duration of prophylaxis and lack of distinction between prophylaxis and therapy among surgical patients; irrational antibiotic prescribing in gastroenteritis; overuse of quinolones and lack of use of penicillin in pneumonia; overuse of quinolones and lack of use of doxycycline and macrolides in genital infections; and overreliance on antibiotics for treating skin and soft tissue infections.Providing a quantitative comparison of antibiotic use rates for suspected infections, using the “focus of infection” approach along with the ATC/DDD methodology, appears appropriate for identifying targets for quality improvement with regards to antibiotic prescribing. Antibiotic resistance is a rapidly increasing public health problem The National Policy for Containment of Antimicrobial Resistance, India in 2011 Disease surveillance projects have been initiated in several countries, and national antibiotic policies have been formulated. These projects, which used the ATC/DDD methodology, include Strama in Sweden Accurate evaluation of antibiotic use can be achieved using patient-level surveillance The study sites were 2 hospitals. One is a 570-bed teaching hospital attached to the RD Gardi Medical College, which is a nonpaying facility located approximately 6 kilometers from the city of Ujjain. The consultants in this hospital have an institutional hospital-based practice and are not allowed to work in private practice. The other hospital is a 330-bed nonteaching hospital located in the city of Ujjain. In this hospital, all services are charged, but the hospital is run on a break-even basis. The consultants in the nonteaching hospital are allowed to work in private clinics outside of their official hours of work. Both hospitals cater predominantly to a rural population from the villages surrounding Ujjain city. In both hospitals, most admissions (91%) in medical and intensive care units are emergency admissions, whereas in the surgical units, both elective and emergency admissions are equally common. Treatment guidelines for infectious diseases have not been implemented in either hospital.All the patients in whom the admitting consultant suspected an infectious etiology at the time of admission or during the hospital stay and for which antibiotic therapy was started were included in the study. The patients admitted for infectious etiologies requiring anti-infective agents other than antibiotics and those treated for tuberculosis were not included.The participating departments or units in both hospitals were the departments of pediatrics, including the neonatal intensive care unit; general medicine, including the medicine intensive care unit; general surgery; obstetrics and gynecology; ear, nose, and throat; and orthopedics. A total 72 physicians out of available 75 from the above-mentioned departments participated in the study, a participation rate of 96%. Participation was voluntary.The sample size calculation was based on a pilot study done in March and May 2007, which estimated that between 80 and 90% of all the admitted patients were prescribed antibiotics. Assuming a prevalence of 80% for antibiotic prescribing and requesting 80% power to detect a difference of 10 percentage points between hospitals for a given focus of infection in a statistical test for the comparison of 2 proportions with a 5% significance level, the required sample size was 108 in each of the focus of infection to be compared. Since, we had 15 foci of infection to be compared the minimum sample size was 15 Χ 108 Χ 2 = 3240 patients.The data collection form included patient details, age, sex, admission ward, dates of admission, discharge, prescribed antibiotic (s) (classified according to ATC), start and completion of antibiotic treatment course, dose per administration, number of doses per day, and route of administration. Any change in the antibiotic prescribed, its dose, or duration was noted. The final diagnosis was coded according to the targeted anatomical systems or subsystems identified as “focuses of infection”  [4], [17The questionnaire was attached to the inpatient files of all the admitted patients. The admitting consultant filled in the diagnosis codes. Each focus of infection was categorized into 1 of 3 indications for therapy or prophylaxis: (a) community-acquired infection; (b) hospital-acquired infection , 1 summer (March to June), and the rainy season (July to October).Each prescribed antibiotic was coded according to the WHO Collaborating Centre for Drug Statistics Methodology, ATC classification index with DDDs 2009 The data were entered into the EpiData Entry (version 3.1) and then transferred to the Stata 10.0 for further analysis . A descriptive data analysis was conducted to understand the frequency of the patient-related variables , foci of infection, and prescribed antibiotic and its class. The Pearson chi-square test was used to test for statistical significance (5%).The ethics committee of RD Gardi Medical College approved the study . Informed written consent was obtained from all the patients. The research was conducted according to the principles expressed in the Declaration of Helsinki. The study did not interfere with the ongoing treatment of the patients.The study included 6026 admitted patients, 2525 (42%) of whom were women and 3501 (68%) were men; 2352 patients (39%) were from the teaching hospital, and 3674 (61%) were from the nonteaching hospital. A total of 5531 patients (92%) were prescribed antibiotics for various foci of infection. The patient's age group, sex, the hospital service, and the season in which the patient was admitted were significantly associated with antibiotic prescribing . Most paIn the teaching hospital, the prescribed antibiotics were ciprofloxacin (72 DDD/HPD), metronidazole (35 DDD/HPD), and cefotaxime (35 DDD/HPD). In the nonteaching hospital, ceftriaxone (40 DDD/HPD), ceftriaxone/sulbactam (4 DDD/HPD), ciprofloxacin (22 DDD/HPD), ofloxacin (20DDD/HPD), and metronidazole (30DDD/HPD) were prescribed .The upper and lower gastrointestinal tracts were the most common sites of infection reported (n = 844) in the general surgery patients. Most of the patients  received prophylactic antibiotic therapy, and 89% received it for more than 24 hours post surgery.In the teaching hospital, nitroimidazoles were the most common antibiotics prescribed , followed by quinolones , tetracycline, and third-generation cephalosporins .In the nonteaching hospital, third-generation cephalosporins  were the commonest antibiotic class prescribed, followed by nitroimidazole , followed by quinolones (42 DDD/HPD-ciprofloxacin 94% and levofloxacin 5%), penicillins with extended spectrum , and third-generation cephalosporins (22 DDD/HPD-cefotaxime 66% and ceftriaxone 24%); co-trimoxazole was also commonly prescribed. In the nonteaching hospital, third-generation cephalosporins , quinolones (32 DDD/HPD-ciprofloxacin 68% and levofloxacin 22%), and co-amoxiclav were prescribed.The prescribing pattern for bronchitis was similar to that for pneumonia in the respective hospitals .In the treatment of renal infections, the antibiotic prescribing pattern was similar in the 2 hospitals. The quinolones, third-generation cephalosporins, and aminoglycosides were the most common classes prescribed. In the nonteaching hospital, however, a greater proportion of levofloxacin, ceftriaxone with a beta-lactam inhibitor, amikacin, and nitelmycin were prescribed .In the patients with genital infections (98% women), higher amounts of DDD/HPD were prescribed in the nonteaching hospital than in the teaching hospital . In the In both hospitals, skin and soft tissue infection (SSI) was most commonly treated with co-trimoxazole . In the teaching hospital, aminoglycosides (58 DDD/HPD-gentamicin 65% and 32% amikacin), third-generation cephalosporins (44 DDD/HPD-cefotaxime 45% and ceftriaxone 34%), nitroimidazoles, and quinolones were prescribed .In the nonteaching hospital, the most common class prescribed was third-generation cephalosporins (92 DDD/HPD-ceftriaxone 36% or ceftazidime with beta-lactamase inhibitor 20%), followed by nitroimidazoles (37 DDD/HPD-metronidazole 69% and tinidazole 22%), aminoglycosides (35 DDD/HPD), and quinolones . The results show that more DDD/HPD were prescribed in the teaching hospital for nearly all the foci of infections. These findings are similar to those of a large French study that demonstrated that higher antibiotic DDDs were dispensed in teaching hospitals Approximately 9% of the infections were classified as HAI, which is a lower incidence than the expected rate of around 25% Approximately one third of all antibiotic prescribing was for prophylaxis, mainly in surgical services. Most (86%) of these patients received antibiotics for longer than 24 hours, which is a cause of concern that should be considered for possible intervention. Unnecessarily long surgical prophylaxis was also reported in the ESAC hospital point prevalence studies in 2006, 2008, and 2009 Variation in antibiotic prescribing according to age groups was observed in this study, with higher rates (by 4 to 9% unit points) of antibiotic prescribing in adults than in children. Such variation was documented previously in the same setting in an outpatient diagnosis prescribing study We analyzed the pattern of antibiotic prescribing in specific foci of infection. Over prescription of antibiotics for diarrhea is a major public health problem in India Prophylactic use of antibiotics is common in patients undergoing surgical procedures Streptococcus pneumoniae and Klebsiella pneumoniae, the 2 organisms most frequently associated with adult pneumonia in the Asia-Pacific region S. pneumoniae from the Asia-Pacific are penicillin resistant (according to an MIC of 8 µg/mL) Doxycycline and tetracycline dominated the prescription pattern for pneumonia in the teaching hospital, and these are appropriate for community-acquired pneumonia (CAP) of all severities according to the British Thoracic Society guidelines The Infectious Disease Society of America recommends the use of 1 of the following 3 initial therapies for acute pyelonephritis in adults: (a) fluroquinolone, (b) aminoglycoside with or without ampicillin, or (c) extended spectrum cephalosporin with or without aminoglycoside Neisseria gonorrhoeae, Chlamydia trachomatis, and Mycoplasma genitalium. Because no single agent covers all of these organisms, combination therapy is recommended N. gonorrhoeae isolates has been documented in the Asia-Pacific region There is a significant unmet demand for the treatment of genital infections among women in rural India due to their high disease burden Among hospital patients with SSI, antibiotic therapy should be initiated only if the patient fails to respond to incision and drainage or shows abscess with severe and extensive disease, rapidly progressive cellulitis, signs and symptoms of systemic disease, associated comorbidities, immunosuppression, or an abscess in an area difficult to drain  The main strength of the present study is that unlike most surveillance Using a data collection procedure that can produce good focus of infection-specific information on ATC/DDD, we were able to identify targets for quality improvement in antibiotic prescribing. The targets identified are higher antibiotic prescribing in a teaching hospital compared with a nonteaching hospital, longer than recommended duration of prophylaxis and lack of distinction between prophylaxis and therapy among surgical patients, irrational antibiotic prescribing in gastroenteritis, overuse of quinolones and lack of use of penicillin in pneumonia, overuse of quinolones and lack of use of doxycycline and macrolides in genital infections, and overreliance on antibiotics in treating skin and soft tissue infections. The study provides much needed Indian data for policy makers to design strategies for promoting prudent antibiotic use and formulating national, regional, and local therapeutic guidelines.


Statement of Purpose. IgG4-related disease (IgG4-RD) is usually associated to an increase of serum IgG4 levels. However other conditions have also been associated to high serum IgG4 levels. Methods. All IgG subclasses analyses performed in our hospital over a one-year period were analyzed. When IgG4 level were over 1.35 g/L, the patient's clinical observation was analyzed and both final diagnosis and reason leading to IgG subclasses analysis were recorded. Only polyclonal increases of IgG4 were considered. Summary of the Results. On 646 IgG subclass analysis performed, 59 patients had serum IgG4 over 1.35 g/L. The final diagnosis associated to serum IgG4 increase was very variable. Most patients (25%) presented with repeated infections, 13.5% with autoimmune diseases, and 10% with IgG4-RD. Other patients presented with cancer, primary immune deficiencies, idiopathic interstitial lung disease, cystic fibrosis, histiocytosis, or systemic vasculitis and 13.5% presented with various pathologies or no diagnosis. Mean IgG4 levels and IgG4/IgG ratio were higher in IgG4-RD than in other pathologies associated to elevated IgG4 levels. Conclusions. Our study confirms that elevation of serum IgG4 is not specific to IgG4-RD. Before retaining IgG4-RD diagnosis in cases of serum IgG4 above 1.35 g/L, several other pathological conditions should be excluded. Immunoglobulin G4 (IgG4) represents the less abundant of the four IgG subclasses in human serum accounting for 3 to 6% of the total IgG .IgG4 has been associated with several pathological conditions. Most of these associations suggest a protective effect of IgG4, such as in allergen-specific immunotherapy  and protIn 2001, Hamano et al. report a quantitative serum IgG4 elevation during sclerosing (or “autoimmune”) pancreatitis above the cutoff value of 135 mg/dL in 95% of patients with autoimmune pancreatitis , leadingFew works have systematically studied diagnosis associated with a serum IgG4 elevation , 16, 17.st, 2009 to December 31th, 2009 at the laboratory of Immunology of our University Hospital of Marseille, France, were analyzed. Serum total IgG and IgG subclasses (1 to 4) levels were measured by immunonephelometry  with reagents from Siemens . All results with serum IgG4 polyclonal increase above the cutoff value of 1.35 g/L were considered for the study. All results for IgG subclasses evaluation performed from January 1P < 0.05. Figures and statistics were realized using GraphPad Prism v. 4.0.The patients' medical records were retrospectively analyzed and demographic, clinical, paraclinical, and evolutive characteristics were recorded. The reason leading the physician to the prescription of IgG subclass evaluation was also recorded. Patients were classified according to the disease associated to IgG4 elevation. Mean values in different groups were compared using the Mann-Whitney test. Results were considered significant for A total of 646 IgG subclass analyses were recorded at the laboratory of Immunology during the one-year period. An IgG4 level above 1.35 g/L was found on 75 samples that corresponded to 60 different patients (some of them took 2 or more samples during the year). Among these 60 patients, data could be collected for 59 patients. sex ratio 1/1). Mean age at IgG subclass measurement was 47.2 years (range: 4–85 years). Mean serum IgG level was 18.50 g/L (range: 7.38–40.4 g/L). Thirty-eight patients (64%) presented an elevated serum IgG level (>14 g/L). By definition, a serum IgG4 level above 1.35 g/L was present in all 59 patients. Mean serum IgG4 level was 4.35 g/L (range: 1.37–20.6 g/L).Among the 59 patients analyzed, 30 were men and 29 were women , Nocardia, Toxoplasma gondii, Enterobius vermicularis) were variable in this group. In 8 patients (13.6%), final diagnosis was an autoimmune disease: systemic lupus erythematosus (SLE) in 4 patients (with secondary antiphospholipid syndrome in 2), Sjögren Syndrome (SS) in 2 patients, Biermer disease in 1 patient, and systemic sclerosis in 1 patient. An IgG4-related disease (IgG4-RD) was the final diagnosis in 6 patients (10.1%). Histological documentation was available in all cases of IgG4-RD with characteristic histopathological features with lymphocytic and plasmacytic polyclonal inflammatory infiltrate  and fibrosis. Organ involvements included sclerosing pancreatitis in 4 patients, tubulointerstitial nephritis in 5 patients, polyadenopathy in 5 patients, sialadenitis in 3 patients, sclerosing cholangitis in 2 patients, dacryoadenitis in 1 patient, hypophysitis in one patient, and inflammatory pseudotumors . Four of these patients with IgG4-RD were under treatment (corticosteroids and/or immunosuppressive therapy) at the time of IgG subclass measurement. In 5 patients (8.5%), an IgG4-RD was considered as possible. These patients presented with one or more compatible organ involvements but without histopathological documentation. Sclerosing cholangitis was presented in 4 cases , sclerosing pancreatitis in the other case. In 4 patients (6.8%) the final diagnosis was a cancer: ampullary carcinoma, angioimmunoblastic T-cell lymphoma, pancreatic carcinoma, and bronchopulmonary carcinoma. Primary immune deficiency (PID) was the final diagnosis in 4 cases (6.8%): Wiskott-Aldrich syndrome, chronic granulomatous disease, common variable immunodeficiency, and complex humoral immune deficiency. Hypogammaglobulinemia on blood electrophoresis was found in 3 of these patients. Idiopathic interstitial pneumonitis was the final diagnosis in 3 patients (5%) (with pulmonary fibrosis in 2 cases). Cystic fibrosis, Erdheim-Chester disease and vasculitis (hepatitis C-virus-associated type-II-mixed cryoglobulinemia in one case and microscopic polyangiitis in another case) were, respectively, diagnosed in 2 patients (3.4%). In 8 patients (13.6%), no final diagnosis could be retained. Final diagnosis in patients who presented with elevated levels of serum IgG4 was divided into different categories presented in Apart from the diagnosis category considered, allergic and atopic manifestations  were found in the record of only 10 patients (16.9%).P = 0.0021), “auto-immune diseases” (P = 0.0127), “absence of diagnosis” (P = 0.0027), “possible IgG4-RD” (P = 0.0173), “cancer” (P = 0.0095), and “primary immune deficiency” (P = 0.0095). Difference with the group “idiopathic interstitial pneumonitis” was not statistically significant (P = 0.1667). Statistical analysis with the groups “cystic fibrosis,” “Erdheim-Chester disease,” and “vasculitis” was not possible because of the too small size of these groups (n < 3).Serum IgG4 levels found in patients of each diagnosis category are presented in ratio was performed. Serum IgG4/serum IgG ratios observed in patients of each diagnosis category are presented in ratio observed in IgG4-RD group was significantly more important than in the groups “repeated infections” (P = 0.0217), “auto-immune diseases” (P = 0.0127), “absence of diagnosis” (P = 0.0426), and “cancer” (P = 0.0381). Differences with the groups “possible IgG4-RD” (P = 0.0519), “primary immune deficiency” (P = 0.1143), and “idiopathic interstitial pneumonitis” (P = 0.2619) were not statistically significant. Statistical analysis with the groups “cystic fibrosis,” “Erdheim-Chester disease,” and “vasculitis” was not possible because of the too small size of these groups (n < 3).An analysis of the serum IgG4/serum IgG Serum IgG4 elevation above 1.35 g/L has been shown to be a predictive marker of type 1 AIP . UsefulnIn IgG4-RD, the Japan G4 team has retained the elevation of serum IgG4 above 1.35 g/L as an individual diagnosis criteria, . In factHowever, IgG4 elevation has been reported in several well-characterized pathologies as pancreatic carcinoma , allergThus, exclusion criteria, including pathologies other than IgG4-RD, should be added to the diagnosis criteria as proposed by recent CDC criteria for IgG4-RD . To bettOnly 10% of 59 patients with elevated serum IgG4 (>1.35 g/L) were diagnosed with IgG4-RD. Other diagnoses associated were infections, auto-immune diseases, cancers, primary immune deficiencies, idiopathic interstitial pneumonitis, or vasculitis. Only patients with IgG subclass evaluation and IgG4 above 1.35 g/L were included in this retrospective study without any control group. Thus, the specificity or the sensitivity of serum IgG4 elevation for diagnosis of IgG4-RD could not be assessed.Mean serum IgG4 value was found significantly more important in the IgG4-RD patients. However, IgG4 values observed were overlapping with values observed in some other groups . As already reported, IgG4 elevation was found to be associated with cystic fibrosis , vasculiPseudomonas aeruginosa (present in our two patients) [Cystic fibrosis has already been associated with serum IgG4 elevation during colonization and infection by atients)  and immeatients) . These oatients) . HoweverIn 3 cases, a diagnosis of idiopathic interstitial lung disease was retained. Different intrathoracic involvements have been reported during IgG4-RD including interstitial lung disease . BecauseAllergic manifestations were noted in only 10 patients (16.7%) in our study and could therefore not account for IgG4 elevation in this population. Our retrospective study clearly confirms that serum IgG4 elevation above 1.35 g/L is not specific for the diagnosis of IgG4-RD and can be observed in several clinical situations. The serum IgG4 elevation is more important in IgG4-RD but associated with an important variability within this group. Further studies are needed to define the sensibility and specificity of IgG4 values for the diagnosis of IgG4-RD. Until more specific biomarkers for IgG4-RD are made available, it must be kept in mind that several pathologies should be evoked end excluded in case of IgG4 elevation, before IgG4-RD diagnosis is retained. IgG4-related disease (IgG4-RD) is characterized by one or several fibroinflammatory organ involvements with typical pathological findings. A serum IgG4 elevation above 1.35 g/L is currently retained as an important biomarker of the disease, included in the diagnosis criteria. We confirm in this retrospective study, analyzing systematically the diagnosis associated with serum IgG4 above 1.35 g/L in a large unselected cohort of patients evaluated for IgG subclass, that IgG4 elevation is not specific of IgG4-RD. However, the patients with defined IgG4-RD presented with the most elevated serum IgG4 levels. Thus, several different pathologies should be excluded before IgG4-RD is retained in the context of serum IgG4 elevation.


Medical emergency teams (MET) are implemented to ensure prompt clinical review of patients with deteriorating physiology with the intention of averting further deterioration, cardiac arrest and death. We sought to determine if MET implementation has led to reductions in hospital mortality across a large metropolitan health network utilising routine administrative data submitted by hospitals to the Department of Health Victoria.The Victorian admissions episodes data set (VAED) contains data on all individual hospital separations in the State of Victoria, Australia. After gaining institutional ethics approval, we extracted data on all acute admissions to metropolitan hospitals for which we had information on the presence and timing of a MET system. Using logistic regression we determined whether there was an effect of MET implementation on mortality controlling for age, gender, Charlson comorbidity diagnostic groupings, emergency admission, same day admission, ICU admission, mechanical ventilation, year, indigenous ethnicity, liaison nurse service and hospital designation.P < 0.001. In a multivariable logistic regression, mortality was associated with a MET team being active in the hospital for more than 2 years. The odds ratio for mortality in hospitals where a MET system had been in place for greater than 4 years duration was 0.90 (0.88-0.92). Mortality during the first 2 years of a MET system being in place was not statistically different from pre-MET periods.5911533 individual admissions and 73,599 associated deaths from July 1999 to June 2010 were included in the analysis. 52.2% were male and median age was 57(42-72 IQR). Mortality rates for MET and non-MET periods were 3.92 (3.88-3.95 95%CI) and 4.56 (4.51-4.61 95%CI) deaths per 1000 patient days with a rate ratio after adjustment for year of 0.88 (0.86-0.89 95%CI) Utilising routinely collected administrative data we demonstrated that the presence of a hospital MET system for greater than 2 years was associated with an independent reduction in hospital mortality across a major metropolitan health network. Mortality benefits after the introduction of a MET system take time to become apparent. Medical emergency teams (MET) were conceived to provide prompt clinical review of deteriorating patients with the intent of averting further deterioration, cardiac arrest and death . The calMET systems rely on a hospital process made up of an afferent limb, an efferent limb and a management team ,8. The aIn Australia, MET teams are medically led and are distinct from critical care liaison/outreach services. The latter are nurse-led services with broad roles and varied responsibilities, which, in addition to the review of deteriorating patients, include coordination of ICU discharge, routine review of ward patients, improving communication between units, and ward education and support . Such seBefore-and-after studies from a number of institutions support the premise that a MET reduces mortality -15, althLarge medical administrative databases are increasingly being used to examine health care quality and the association between interventions and patient outcomes ,21. In VThe study was undertaken at St Vincent's Hospital Melbourne, Victoria, Australia, and ethics approval for the study was obtained from the Hospital Research and Ethics committee of St Vincent's Hospital Melbourne.Each year the Department of Health Victoria distributes a de-identified subset of the VAED to each Victorian Hospital. The data contain demographic details, reasons for and type of admission to hospital as well as up to 40 diagnostic (ICD-10-AM) and 40 procedure codes. We used these files to extract data for a 10-year period on all acute admissions to public hospitals, for which we had information on the presence and timing of a MET system in a metropolitan area, including the state capital Melbourne and a large satellite city, Geelong. The combined population of these cities was approximately 4,255,000 in June 2010 and their combined health services also provide specialised services for the state of Victoria, which has a total population of about 5.5 million. Data on the timing and presence of a MET team and ICU liaison nurse outreach service was determined by contacting the Directors of Intensive care at the relevant hospitals. Information on the nature of the teams, their calling criteria and oversight and audit processes was not determined. A categorical variable for MET was defined as the absence of MET and the presence of MET for < 2yrs, 2 to 4 years or > 4 years. The presence or absence of an ICU liaison service was included as a binary variable.The data in the VAED is de-identified. Variables for age, gender, ICD-10-AM codes, admission hospital, length of stay, ICU admission, mechanical ventilation, financial year of admission and indigenous ethnicity were extracted. These diagnostic codes were used to determine the comorbidity elements of the Charlson comorbidity index  as a meaP-values < 0.05 were considered significant. Statistical analysis was performed using Stata 12 .Continuous data were summarised as mean ± SD if approximately normally distributed or median and interquartile range (IQR) if skewed. Estimates are shown with 95% confidence intervals (CI). Categorical variables were reported as counts and proportions with differences assessed by the chi-square and Fisher's exact test as appropriate. Mortality rates per 1,000 inpatient days were calculated and Mantel-Haenszel methods used to determine rate ratios. Logistic regression was used to determine whether there was an independent association of MET implementation with mortality. From July 2000 to June 2010 there were 5,911,533 individual admissions and 73,599 (1.25%) deaths; 3,375,923 (57.1%) were coded as same-day admissions. Among the total admissions, 52.2% of patients were male and the median age was 57 (IQR 42 to 72) years. The patients came from twelve hospitals, six of which were designated tertiary and six metropolitan. The median number of admissions per hospital per year was 42,788 . One of the hospitals had an active MET at the beginning of the study period and by the end of the study period all but three hospitals had an active MET. The percentage of patients exposed to a MET increased from 5.3% at the commencement of the study to approximately 80% of patients in the final 4 years  per 1,000 patient days. Unadjusted mortality decreased in association with the introduction of a MET (Table P < 0.001).Univariable analysis demonstrated an association between mortality and the presence of a MET, the presence of a liaison service, year, age, ICU hours, mechanical ventilation, emergency admission, hospital level and Charlson diagnostic groups (results not shown). These variables were included in a multivariable logistic regression model. In the multivariable model, mortality was associated with patient age, year of admission, gender, ICU admission, mechanical ventilation, hospital designation, emergency admission, multiday admission, Charlson comorbidity indices and the presence of a MET team for 2 or more years  orders. Both may result in a reduction in cardiac arrest calls but only the former will lead to a reduction in hospital mortality. Furthermore, MET may have other benefits such as preventing deterioration and complications, preventing ICU admissions, ward education and staff satisfaction , and 'chThis study is limited by its retrospective nature, dependence on reliable coding across many years and institutions, and the limited data with which to adjust for individual patient differences. Trained data extractors collect VAED data and the information is used to code all hospital separations. It is submitted to the Department of Health Victoria and forms the basis of funding for Victorian public hospitals. Furthermore, it is internally and externally audited at regular intervals to assess completeness and accuracy. Its use for the study can be criticised for not being purpose-collected, however, it does contain parameters that are known to be associated with outcome and we have previously shown that use of these data yields similar results to analysis of prospective purpose-collected data .Considerable changes in health systems will have occurred concurrently with the introduction of MET, including critical care liaison/outreach. The duties of critical care outreach teams overlap with that of MET and may be additive to any outcome benefit associated with MET. Studies of ICU outreach have shown variable results but they have been associated with reductions in adverse events, ICU readmission and mortality, as well as improved nursing confidence and knowledge . Given tThe Hosmer-Lemeshow goodness of fit-statistic for the logistic model suggested poor calibration. However, Kramer and Zimmerman  studied Using routinely collected administrative data we have shown that the implementation of MET systems at individual hospitals was associated with a reduction in hospital mortality across a large metropolitan health system. This effect took time to become apparent, consistent with the assumption that the MET system takes time to alter hospital culture and process. Given that randomised trials of MET are unlikely to occur in the future, administrative data may provide a useful tool for building the evidence base for MET both at institutional and health network levels.• Mortality across a large metropolitan health service decreased in association with the introduction of medical emergency teams• Change in mortality took time to become apparent• Delayed reduction in mortality is consistent with MET implementation taking time to change hospital process and culture• Routinely collected government administrative data may be useful to monitor changes to health service processCI: confidence interval; IQR: interquartile range; MET: medical emergency team(s); NFR: not for resuscitation; OR: odds ratio; VAED: Victorian admissions episodes dataset.The authors declare that they have no competing financial or non-financial interests.AT performed the statistical analysis and drafted the manuscript. JS conceived the study concept, extracted and cleaned the data and helped draft the manuscript. Both authors have read and approved the manuscript for publication.


Dot arrays are often used to study basic numerical skills across cultures, species and development. Researchers investigate the ability of subjects to discriminate between dot arrays, as a function of the ratio or distance between their numerosities. Such studies have contributed significantly to the number sense theory —possibly the most influential theory in numerical cognition literature today. However, a dot array contains, in addition to numerosity, continuous properties such as the total surface area of the dots, their density, etc. These properties are highly correlated with numerosity and therefore might influence participants' performance. Different ways in which different studies choose to deal with this confound sometimes lead to contradicting results, and in our opinion, do not completely eliminate the confound. In this work, we review these studies and suggest several possible reasons for the contradictions in the literature. We also suggest that studying continuous properties, instead of just trying to control them, may contribute to unraveling the building blocks of numerical abilities. Humans and animals share the ability to estimate and discriminate magnitudes. In animals, these abilities are crucial for survival. For example, swimming with the larger shoal increases the probability of fish to survive , one can only estimate continuous properties, even without time limitation. For example, no matter how much time you have, you cannot tell the exact cumulative area of the dots in an array. You can only estimate it. This review will deal mainly with visually presented non-symbolic quantities.numbers. A “number processor” component allows both humans and animals to “represent quantities and transform them according to the rules of arithmetic” (p. 18). The ANS theory . Note that the majority of studies involving dot arrays focus on numerosities; the numerosity is systematically manipulated and the participants' attention is directed to this dimension. Furthermore, as mentioned in a review by Mix et al.  should be the numerosity, and continuous properties  should be identical. This is physically impossible. For example, if there are four squares in one array and 2 in another array and all the squares are of the same size, the four squares have greater total surface area than the two squares. To control for area, one can equalize the total surface of the two arrays. However, this means that the density and average size will have changed  is enough to make a decision. For example, fish select a shoal by comparing the sizes of two shoals. It is impossible to determine that their choice was based on the absolute number of fish in a shoal. In contrast, using numerosities might help when one needs to produce an exact value. For animals, this is rarely the case. If animals need to enumerate, it is usually in the subitizing range   to evade the above mentioned problems. However, it seems that other dimensions are susceptible to similar difficulties. For example, Beran  had monkTo summarize, continuous and discrete properties are highly correlated. Consequently, it is hard to exclude the influence of continuous properties and study the influence of a discrete property—numerosity—in isolation. Studies used various methods to control the effects of continuous properties. Those studies worked under the assumption that the experimental design prevented participants from using continuous properties. However, in most of those studies, this assumption was not verified. There was either no attempt to test for possible influence of continuous magnitudes, or, like in Odic et al.'s  study, oRelying on continuous magnitudes enables “quick and dirty” estimations; for example, where there are more predators, where there is more food, etc. From the ATOM point of view, the faster we make magnitude estimations, the faster we will be able to plan and execute actions. From an evolutionary perspective, it is sufficient in most cases to make “quick and dirty” estimations. Hence, it makes sense to theorize that if we are born with the ability to discriminate magnitudes, it is due to discriminating continuous and not discrete properties.Von Aster and Shalev  suggesteOur suggested model is illustrated in Figure We reviewed studies investigating comparative judgment of dot arrays and showed the contradicting results they have found. We suggest that these conflicting results are due to the fact that it is physically impossible to completely neutralize the effect of continuous properties, not only in the visual domain. The different ways that different studies chose to deal with this confound has led to variability in the results and the conclusions. Thus, in our opinion, theories such as the number sense and the ANS, which consider numerosity to be the most salient cue and the basis for our numerical abilities, should be revised to include continuous properties. We further suggest that directly studying the contribution of continuous properties to the ability to discriminate magnitudes, throughout normal and impaired development, in adults as well as in children, might shed light on the basis of numerical cognition.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Systematic reviews are a cornerstone of evidence-based medicine but are useful only if up-to-date. Methods for detecting signals of when a systematic review needs updating have face validity, but no proposed method has had an assessment of predictive validity performed.The AHRQ Comparative Effectiveness Review program had produced 13 comparative effectiveness reviews (CERs), a subcategory of systematic reviews, by 2009, 11 of which were assessed in 2009 using a surveillance system to determine the degree to which individual conclusions were out of date and to assign a priority for updating each report. Four CERs were judged to be a high priority for updating, four CERs were judged to be medium priority for updating, and three CERs were judged to be low priority for updating. AHRQ then commissioned full update reviews for 9 of these 11 CERs. Where possible, we matched the original conclusions with their corresponding conclusions in the update reports, and compared the congruence between these pairs with our original predictions about which conclusions in each CER remained valid. We then classified the concordance of each pair as good, fair, or poor. We also made a summary determination of the priority for updating each CER based on the actual changes in conclusions in the updated report, and compared these determinations with the earlier assessments of priority.The 9 CERs included 149 individual conclusions, 84% with matches in the update reports. Across reports, 83% of matched conclusions had good concordance, and 99% had good or fair concordance. The one instance of poor concordance was partially attributable to the publication of new evidence after the surveillance signal searches had been done. Both CERs originally judged as being low priority for updating had no substantive changes to their conclusions in the actual updated report. The agreement on overall priority for updating between prediction and actual changes to conclusions was Kappa = 0.74.These results provide some support for the validity of a surveillance system for detecting signals indicating when a systematic review needs updating. Systematic reviews are a cornerstone of evidence-based care, either by themselves or through their incorporation into practice guidelines, performance measures or other evidence-based practice. To be useful, however, systematic reviews need to be up-to-date.The science of determining when systematic reviews need updating has been developing for the past decade. Prior to 2001, no method or criterion existed to determine whether evidence-based products remained valid or whether the evidence underlying them had been superseded by newer work. Since then, several groups have begun developing methods to determine signals for updating reviews . Most meet al.[In this study, we assessed the predictive validity of signals for updating CERs detected in 2009 that have since been updated. We start with a description of the original process used to detect signals  and thenet al.; the twoSearch strategy. We started by using the search strategy employed in the original report. However, we limited the search  to five top-rated general interest medical journals  and the specialty journals most relevant to the topic. The specialty journals were those most highly represented among the references from the original report . We also modified the key terms if, for example, we were aware of new drugs for the condition, adding their names to the search terms. Search inception dates were 6 to 12 months prior to the end date of the original CER search in order to ensure overlap between the searches.Study selection and extraction. Using the same general inclusion and exclusion criteria as the original CER, a single reviewer experienced in systematic reviews conducted a screening of the titles and abstracts and requested any articles deemed relevant to the topic. From among those articles, the reviewer extracted relevant data from articles that met the inclusion criteria and then constructed an evidence table. These data included study-level details extracted in the original CER  as well as the outcomes themselves.Identifying new evidence from experts and expert opinion. For each topic, we created a questionnaire matrix that listed the key questions and conclusions from the original executive summary. The matrix was sent to experts in the field, including the original project leader, technical expert panel members and peer reviewers. The experts were asked to indicate whether each conclusion listed in the matrix was, to their knowledge, still valid and, if not, to describe any new evidence and provide citations.Assessing individual conclusions for signals. Once abstraction of the study conditions and findings for each new included study was completed and expert opinions were received, we assessed, on a conclusion-by-conclusion basis, whether the new findings provided a signal for the need for an update. Table For each CER, we constructed a summary table that included the following for each key question: original conclusions, findings of the new literature search, summary of expert assessment, our final assessment of the currency of the conclusions, and the priority for updating.Determining priority for updating a CER. We needed to make an overall judgment regarding the priority for updating an entire CER. This determination rested on two criteria. (1) How much of the CER is possibly, probably or certainly out-of-date? (2) How out-of-date is that portion of the CER? For example, we asked whether the potential changes to the conclusions would involve only refinement of original estimates or whether the potential changes would include the finding that some therapies are no longer favored or might no longer be in use. Another question was whether the portion of the CER that was probably or certainly out-of-date involved an issue of safety  or the availability of a new drug within an existing class, with the latter being a less important signal to update than the former. This final determination was a global judgment made by all the individuals working on each particular CER. On the basis of that determination, we classified CERs as being of low, medium or high priority for updating. For high-priority updates, we also provided our rationale.Our 2009 work assessed 11 CERs. We classified four as having a high priority for updating, four as having a medium priority for updating and three as having a low priority for updating  and the search dates of the update reports (2010 to 2012). Therefore, for conclusions with poor concordance, we reviewed whether they may have been influenced by new evidence published after the surveillance signals search.We performed our assessment of predictive validity for nine CERs comprising 149 individual conclusions. For each CER, we present our assessment of the concordance of individual conclusions  of conclusions for each CER and across CERs had good concordance. However, the CER on gastroesophageal reflux disease (GERD) had four “out-of-date” conclusions with only fair concordance, and one conclusion we had assessed as “still valid” was shown to be out-of-date.The published 2009 updating assessment judged that the conclusion regarding endoscopic treatment for GERD “should be deleted”, meaning that it was out-of-date, because the endoscopic procedures had been withdrawn from the market. However, one of the three endoscopic procedures reviewed in the original report continued to be used, new endoscopic procedures were introduced and one of the two withdrawn procedures was later reintroduced. The update report noted this changing landscape, and we deemed the concordance with the 2009 prediction as only fair. A more appropriate surveillance assessment would have been that the conclusion needed updating because the endoscopic procedures were evolving over time.Another conclusion in the original GERD report—that surgery and medical therapy were similarly effective—was rated as “still valid” during the surveillance process but had poor concordance with the update review, which concluded that surgery was favored over medical therapy. One of the studies providing new evidence in support of this conclusion was published in 2009, after completion of the surveillance signal search.Table This assessment of the predictive validity of a method to assess a CER for signals for updating yielded generally favorable results. For the vast majority of individual conclusions, concordance between the 2009 predictions and the subsequent updated conclusions was judged to be good. The one instance of poor concordance had new evidence published after the surveillance signals had been assessed, and in this instance involved a CER already judged to be of high priority for updating based on signals of other out-of-date conclusions.Our present study has three primary limitations. The first is sample size. We were able to assess only nine CERs. However, this number included CERs assessed as being of high, medium or low priority, thus allowing us to assess the possibility of false-negatives . The likelihood of assessing such false-negatives again is small, as it requires that low-priority CERs be subjected to the gold standard of a full update. Our findings that neither of the CERs judged to be a low priority had any substantive changes in conclusions will reinforce the decision to invest scarce resources in researching other topics rather than commisioning updates of low-priority CERs.A second limitation is the matching of original conclusions to updated conclusions. In some updated reports, the authors themselves matched the conclusions. In most cases, however, this was not done, and, in some circumstances, determining the appropriate match to the original conclusion was challenging. Additional file The third principal limitation of this study is that the 2013 assessment of the 2009 predictions could not be made in a blinded fashion. Our Evidence-based Practice Center (EPC) did both assessments, and, even if some other group had done the 2013 assessment, we could not have enforced blinding, because the 2009 assessments are in the public domain. We tried to guard against bias by having explicit reasons for each judgment and presenting these reasons for readers themselves to judge. Our reasoning should be transparent.With the limitation of small sample size in mind, we offer the following preliminary conclusions about the surveillance signal method. (1) Low-priority CERs are unlikely to have any substantive changes in conclusions. (2) Conclusions judged likely to be “still valid” almost certainly are still valid. (3) Conclusions judged to be “out-of-date” almost certainly are out-of-date. (4) Safety concerns and the appearance of new classes of therapies and more efficacious treatments are the best targets for high-priority updates. (5) The classification of individual conclusions as possibly or probably out-of-date owing to new evidence may be slightly too sensitive as a signal; in a number of such instances, the update report’s conclusion did not change, because the new evidence identified in the signal search was either rejected or insufficient to change the original conclusion.In sum, our assessment provides some support for the predictive validity of this method of assessing CERs for signals of the need for updating. Future research is likely to be confined to assessing updates of systematic reviews judged to be a medium or high priority for updating. Further assessment of the factors leading to changes in individual conclusions may help refine the criteria for distinguishing between high- and medium-priority update topics. However, investing extra time and effort to distinguish “possibly” from “probably” out-of-date conclusions or to further refine the global assessment to distinguish medium- from high-priority update topics may begin to make the surveillance process resemble the actual update, which is not the goal of surveillance. In this application, the surveillance process worked very well—nearly perfectly, in fact (κ ≥ 0.8 is considered nearly perfect agreement). No low-priority CER was judged, as having had a substantive change to a conclusion in the update, whereas 3 of 4 high priority CERs did have substantive changes to the conclusions. The results suggest that it is very unlikely that new, practice-changing evidence exists concerning a systematic review judged to be a low priority for updating and supports a policy of delaying an update of a systematic review until new evidence is sufficient to warrant assigning it at least a medium priority.et al. [The assessment method described herein represents part of the basis for the surveillance method used to assess AHRQ systematic reviews as described by Ahmadzai et al. . That prIn our present study, we found evidence supporting the predictive validity of a method for assessing AHRQ systematic reviews regarding their need for updating. One advantage of this method relative to other proposed methods is that it is equally useful for meta-analytic reviews and narrative reviews. It may be applicable to systematic reviews produced by other organizations.The authors declare they have no competing interests.PGS developed the idea for the study. PGS and SJN developed the original 2009 method and its applications. AM and BJ collected the information from the updated reports and performed the initial matching of conclusions and PGS revised these matches, made the determinations of agreement and concordance, and performed the statistical analysis. All authors read and approved the final manuscript.Concordance of predicted and actual conclusions for update of the nine Comparative Effectiveness Reviews. The table presents the authors assessment of the concordance of individual conclusions for each of the nine comparative effectiveness reviews by listing the amount of conclusions from the report that that were “still valid”, “possibly out of date”, “probably out of date”, “out of date”, or were “not applicable/no matching conclusions/new conclusions” to those that were rated as “good”, “fair”, “poor”, or “not rated”.Click here for fileConclusion assessments across all nine Comparative Effectiveness Reviews. The table presents the nine Comparative Effectiveness Reviews conclusions for the original review, the update review, the 2009 prediction, and the concordance for each of the conclusions.Click here for file


We correlated each perfusion condition (heroin and placebo) separately with a VBM sample that was identical for the two treatment conditions. It was assumed that heroin-associated perfusion is manifested in short-term effects, while placebo-associated perfusion is more related to long-term effects. In order to restrict our analyses to fronto-temporal regions, we used an explicit mask for our analyses. Correlation analyses revealed a significant positive correlation in frontal areas between GM and both perfusion conditions (heroin and placebo). Heroin-associated perfusion was also negatively correlated with GM in the inferior temporal gyrus on both hemispheres. These findings indicate that, in heroin-dependent patients, low GM volume is positively associated with low perfusion within frontal regions.Structure and function are closely related in the healthy human brain. In patients with chronic heroin exposure, brain imaging studies have identified long-lasting changes in gray matter (GM) volume. More recently, we showed that acute application of heroin in dependent patients results in hypoperfusion of fronto-temporal areas compared with the placebo condition. However, the relationship between structural and cerebral blood flow (CBF) changes in heroin addiction has not yet been investigated. Moreover, it is not known whether there is any interaction between the chronic structural changes and the short and long-term effects on perfusion caused by heroin. Using a double-blind, within-subject design, heroin or placebo  was administered to 14 heroin-dependent patients from a stable heroin-assisted treatment program, in order to observe acute short-term effects. Arterial spin labeling (ASL) was used to calculate perfusion quantification maps in both treatment conditions, while Voxel-Based Morphometry (VBM) was conducted to calculate regional GM density. VBM and ASL data were used to calculate homologous correlation fields by Biological Parametric Mapping (BPM) and a whole-brain Pearson  Heroin dependence is associated with chronic exposure to heroin and is characterized by compulsive drug use despite negative consequences, including social drift  . CognitiStructural magnetic resonance imaging (MRI) studies on the long-term effect of opioid application have demonstrated that opioid dependent subjects showed decreased gray matter (GM) in the bilateral prefrontal cortex and bilateral temporal cortex, including the insula . FurtherStudies on cerebral blood flow (CBF) in heroin addiction have showed that chronic heroin use is associated with a decrease in global perfusion, with the greatest decrease in the frontal, occipital, and temporal regions . In our However, the relationship between GM volume and perfusion in heroin addiction has not yet been investigated. In the current study, we examined whether there was an association between GM volume and perfusion in 15 heroin-addicted patients. Perfusion was assessed during the normal drug free condition (placebo) and after administration of heroin. We applied Biological Parametric Mapping (BPM) to assess the relation between perfusion – as measured by arterial spin labeling (ASL) – and GM volume – as measured by Voxel-Based Morphometry (VBM). The BPM toolbox was developed by Casanova and colleagues in MATLAB . This inhttp://clinicaltrials.gov (ID NCT01174927). After receiving a written and oral description of the study aims, all participants gave their written informed consent before inclusion.The study was approved by the local ethics committee and registered under Fourteen  non-left-handed heroin-dependent out-patients were recruited from the Center of Substance Use Disorders of the Department of Psychiatry in Basel University. The inclusion criteria were as follows: age older than 18 years, current heroin-maintained treatment for at least 6 months, with an unchanged heroin dose during the past 3 months. The exclusion criteria were a positive alcohol breathalyzer test, or an additional physical disease or psychiatric disorder other than substance dependencies. Clinically experienced psychiatrists conducted the Structured Clinical Interview for DSM-IV Axis II Disorders (SCID-II)  to assesSubjects reported their age of first heroin use (mean = 18.4 ± 2.9 years), years of heroin dependence (mean = 21.4 ± 7.2 years), duration of heroin maintenance (mean = 7.2 ± 3.9 years), and daily heroin dose (mean = 352 ± 178 mg). Patients were told to abstain from illicit drug use other than the prescribed heroin for the duration of the study, from alcohol intake for 72 h and from tobacco consumption for 2 h before scanning. Illicit substances and medications were controlled by a urine test at each session. Nevertheless, three patients were tested positive for cannabis and six patients for cocaine at one or both points of the measurement.In a cross-over, double-blind design, placebo , and half of the daily heroin dose (mean = 176 ± 89.1 mg) were administered intravenously over a period of 30 s by a study nurse 20 min before the scanning session started. Heroin was dissolved in 5 ml of sterile water and aspirated into a syringe, according to the procedure described by Stohler et al. . Each paScanning was performed on a 3T MRI scanner , using a 3D T1-weighted sequence (MPRAGE) for high resolution anatomical data and an ASL sequence  for quanCerebral blood flow (perfusion maps) was calculated by in-house software from ASL DICOM data. The difference images of the label and control images were first calculated and the time course was fitted to an ideal flow model. The equation for perfusion was then solved . The resVoxel-Based Morphometry Structural data (MPRAGE) were preprocessed with VBM implemented in SPM8. MPRAGE images were non-rigidly normalized to a population-based average using diffeomorphic anatomical registration through exponentiated lie algebra (DARTEL) . The segp < 0.01, without correction (cluster-forming threshold) . Statistical significance was assessed at cluster-level, using non-stationary random field theory . The firreshold) . FinallyFor an integrative analysis of multimodal imaging data, we used BPMp < 0.01. Significant clusters were corrected for multiple comparisons with a FWE of p < 0.05  was correlated separately with the VBM data. Cluster-level inference was performed using a homologous correlation field  and a clp < 0.05 . MNI coor correlations between perfusion and VBM data for each voxel within the standardized MNI space. We used the image calculation function implemented in SPM8 and applied the Pearson formula xi and yi represent perfusion and VBM values, and n = 14).In addition, we calculated Pearson Comparison of the heroin and placebo conditions found no significant difference in either direction (heroin > placebo and heroin < placebo).r values within the cluster maxima were all r > 0.7. No significant cluster was found in the temporal lobe and there was no significant negative correlation between modalities . We also found that heroin-associated perfusion correlated negatively with GM in the inferior temporal gyrus on both hemispheres. This negative correlation is difficult to explain in terms of our hypothesis that hypoperfusion is the driving force leading to GM reduction. The cuneus serves as visual processing and inhibitory control centers and electroencephalographic studies have revealed abnormalities in heroin-dependent individuals , 32 and The regions showing a positive correlation between perfusion and GM are in line with regions showing reduced GM in heroin dependence , 34, 35.The frontal cortex is known to play an essential role in heroin addiction. In a recent study, we could show that heroin impairs stimulus-driven attention allocation, as indicated by reduced activity in the right inferior frontal gyrus . FurtherHowever, the question of how perfusion and GM interact in heroin dependence was not answered in our analysis. A possible explanation could be that heroin-associated perfusion in prefrontal and extended limbic areas may alter cerebral volume due to mild but chronic hypoxia and ischemia. This hypothesis is in line with animal models of brain ischemia, which showed that reduction in perfusion leads to neural death and cognitive impairment , 47. It There are some limitations to this study, which should be carefully considered in interpreting our findings. Firstly, we only used a moderate number of subjects. A second limitation is the lack of a control group. It was thus not possible to analyze regional GM and perfusion differences with a between group approach and it therefore remains unclear how placebo-associated perfusion, reflecting normal perfusion in heroin-dependent patients, differs from that in healthy controls. A further difficulty is the aim of separating pure heroin effects on perfusion and structure from effects of other substances. Abstinence from tobacco use may induce craving, resulting in increased perfusion in areas associated with cigarette craving . HoweverIn conclusion, we demonstrated that local GM volumes at baseline in heroin-dependent patients predicted perfusion in frontal regions after administration of placebo and heroin. These findings may suggest that recurrent and chronic hypoperfusion induced by heroin is a driving force for reduced GM in heroin addiction.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bohdan Nosyk and Julio Montaner argue that the cost-effectiveness of HAART roll out has been significantly underestimated because economic analyses haven't yet taken into account the beneficial impact of HAART on HIV transmission. In 2010 for the first time there was a plateau in the growth of the AIDS epidemic, but the current global financial crisis and drop in AIDS funding are threatening the sustainability of this achievement.Growing evidence suggests that HAART is not just life-saving for those with HIV, but also a highly effective means of preventing HIV transmission. Despite this evidence, there is lingering ambivalence about the expansion of HAART coverage.In this Essay we argue that the cost-effectiveness of HAART roll out has been significantly underestimated, as economic analyses have thus far not considered the secondary benefits of HAART, chief among them the impact of HAART on HIV transmission.We argue that the strategic value of expanded HIV testing and expansion of HAART coverage has dramatically increased. This has opened the door for the possibility of wide-scale implementation of “Seek, Test, Treat and Retain” programs as a means to control HIV- and AIDS-related morbidity, mortality, and transmission at once.The global AIDS pandemic has taken a major toll worldwide whether it is in terms of human suffering, lives lost, or economic impact. At the end of 2009, an estimated 33.3 million people were living with HIV/AIDS, with a total of 24 million accumulated AIDS-related deaths worldwide and 2.6 million new infections in 2009 alone For the first time, in 2010, there has been a plateau in the growth of the epidemic The preventive value of HAART has now been definitively proven in the context of serodiscordant couples, where the immediate use of HAART by the partner with HIV has led to a relative reduction of 96% in the number of linked HIV-1 transmissions, as compared with delayed therapy in a landmark randomized trial HAART has long been deemed to be a cost-effective intervention, as it is effective in reducing morbidity and mortality and increasing life-years and quality-adjusted life years (QALYs) gained As we move forward, additional endpoints will need to be captured to fully understand the economic impact of HAART expansion. For instance, HAART is now accepted as a highly effective means to prevent tuberculosis While challenges in implementation of early detection programs exist


The following funding source was omitted from the published article: The Multicenter AIDS Cohort Study is funded by the National Institutes of Health grant NIH 2 U01 AI035039-22.


Feline Immunodeficiency Virus (FIV) is a viral pathogen that infects domestic cats and wild felids. During the viral replication cycle, the FIV p15 matrix protein oligomerizes to form a closed matrix that underlies the lipidic envelope of the virion. Because of its crucial role in the early and late stages of viral morphogenesis, especially in viral assembly, FIV p15 is an interesting target in the development of potential new therapeutic strategies.Our biochemical study of FIV p15 revealed that it forms a stable dimer in solution under acidic conditions and at high concentration, unlike other retroviral matrix proteins. We determined the crystal structure of full-length FIV p15 to 2 Å resolution and observed a helical organization of the protein, typical for retroviral matrix proteins. A hydrophobic pocket that could accommodate a myristoyl group was identified, and the C-terminal end of FIV p15, which is mainly unstructured, was visible in electron density maps. As FIV p15 crystallizes in acidic conditions but with one monomer in the asymmetric unit, we searched for the presence of a biological dimer in the crystal. No biological assembly was detected by the PISA server, but the three most buried crystallographic interfaces have interesting features: the first one displays a highly conserved tryptophan acting as a binding platform, the second one is located along a 2-fold symmetry axis and the third one resembles the dimeric interface of EIAV p15. Because the C-terminal end of p15 is involved in two of these three interfaces, we investigated the structure and assembly of a C-terminal-truncated form of p15 lacking 14 residues. The truncated FIV p15 dimerizes in solution at a lower concentration and crystallizes with two molecules in the asymmetric unit. The EIAV-like dimeric interface is the only one to be retained in the new crystal form.The dimeric form of FIV p15 in solution and its extended C-terminal end are characteristic among lentiviral matrix proteins. Crystallographic interfaces revealed several interactions that might be involved in FIV replication. Further studies are needed to better understand their biological relevance in the function of FIV Gag during viral replication. Retroviridae, genus lentivirus, which also contains the human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV) and simian immunodeficiency virus (SIV). FIV infection induces an immunodeficiency syndrome in infected domestic cats or wild felids, which resembles HIV-induced AIDS. Therefore, this syndrome has been dubbed feline AIDS. FIV is an important veterinary issue as it infects up to 30% of domestic cats in some parts of the world with apparently no evolution of host resistance [The Feline Immunodeficiency Virus (FIV) is an enveloped virus from the family sistance ,2. Moreosistance . Howeversistance , and thesistance ,7.Matrix protein (MA), p15 in FIV or EIAV and p17 in HIV or SIV, is the N-terminal subunit of the retroviral Gag polyprotein. This Gag polyprotein is involved in the architecture of the viral particle, which is common to HIV and FIV and is essential for infectivity of the virus -10. The After the budding of virus particles from host cells, the Gag polyprotein is cleaved by the viral protease into its different subunits during maturation . In the In the HIV model, the N-terminal myristoyl group of MA can adopt sequestered and exposed conformations  consisteet al. suggested that the dimeric conformation observed for EIAV p15 could correspond to a weakened membrane-binding state and proposed a new alternative membrane-binding mechanism [Previous studies have determined the three-dimensional structure of HIV p17, SIV p17 and EIAV p15 by nuclear magnetic resonance spectroscopy (NMR) ,28-30 anechanism . The comTo characterize the FIV p15 structure, we over-expressed FIV p15. This allowed us to perform the first studies on the oligomeric state of the FIV p15 protein and to determine the crystal structure of full-length FIV p15. The structure of a C-terminal-truncated FIV p15 was solved in turn to assess the role of this region in the oligomerization process. In conclusion, we discuss the conformational differences observed between FIV p15 and the matrix protein of other lentivirus. In addition, we also discuss the possible role of the flexible C-terminus of FIV p15 in the replication cycle.3). These experiments demonstrated that the FIV p15 protein in 50 mM sodium phosphate pH 7.4 was mainly monomeric, whereas a dimeric state was observed for the protein in 50 mM MES pH 6   as the search model [work of 19% (Rfree = 25.5%) , which give rise to the close packing of adjacent helices typical for retroviral matrix proteins Figure A. The thAn analysis of the electrostatic potential of FIV full-length p15 revealed a highly basic platform, with helix h1 having amphipathic character (residues Arg7 to Cys16) Figure B. NumeroFIV p15, despite having a low sequence similarity with HIV, EIAV and SIV MA  is strikingly similar in structure to these lentiviral matrix proteins and can be superposed to each with a root mean square deviation (RMSD) of 1.9 Å, 2.5 Å, and 2.1 Å between Cα pairs, respectively Table . The C-tThere are 6 conserved residues between the proteins  implicates the conserved residue Trp37, localized at the center of helix h2 and the C-terminal end of a contacting p15 monomer (Gly130 to Pro132)  and their 2-fold symmetry related counterparts  implicates an antiparallel assembly made up by residues of the C-terminal end via a 2-fold symmetry axis Figure B. Its av2 per monomer. The interface 3 resembles the interface of EIAV dimers [The third interface (interface 3) involves an interaction between loop l3 and helix h5 from one monomer with loop l3 and helix h4 from the other monomer  from the full-length protein (P21212), and 2 monomers are present in the asymmetric unit . Cross-link experiments showed that this mutant was still able to generate dimers in solution in MES buffer Figure A. Becausr Figure B. This dr Figure B. On ther Figure B was assTo our knowledge, this study is the first to characterize the oligomeric forms of FIV p15, using a combination of dynamic light scattering (DLS), chemical cross-linking, and SEC-MALLS experiments. Our results demonstrate for the first time that the oligomeric state of FIV p15 is dependent both on protein concentration and on the buffer used. The dimerization of FIV p15 protein at high concentration and under acidic conditions is different from the reported trimerization of HIV p17 .in cellulo.Here, we provide evidence that the oligomeric state of the non-myristoylated FIV p15 protein is modulated by pH. The monomeric protein shifts to a dimeric form when the pH decreases. This dimer was stable as demonstrated by its dissociation constant around 10 μM as measured by ITC. These results support the hypothesis that deprotonation, by increasing the pH to 7.4, inhibits the formation of oligomers for FIV p15, as previously observed in the case of myristoylated HIV p17 . Intracein vitro. In the case of HIV, it is thought that the Gag polyprotein is primarily monomeric at low concentrations and is therefore largely cytoplasmic because of the sequestration of the myristoyl group [The oligomeric state of the FIV p15 protein is also concentration-dependent yl group . As its yl group . Howeveryl group ,44, indiyl group . This meAnother feature revealed by the docking experiments is the possibility for the myristoyl group to bind in a groove at the surface of FIV p15. This groove involves residues Asp8, Ala12, Glu55, and Leu95, which are highly conserved between FIV strains. Moreover, this groove is also present in the structure of the HIV MA protein . It is nWe have determined the crystal structure of the FIV p15 full-length protein and of a form that lacks its C-terminal end (p15-Δ120). Both structures are similar, with a RMSD of 0.5 Å between Cα pairs. Like the MA proteins of other lentivirus, the FIV p15 protein has five α-helices. However, these helices have different lengths compared to the published structures of the lentiviral MA proteins for HIV , SIV 3232, and EAs stated above, we were able to observe the complete, extended C-terminal end of the full-length FIV p15 due to crystal contacts between Pro131 and Trp37 of the complementary monomer. One cannot exclude a possible influence of the C-terminal histidine tag on the flexibility that we observe for the C-terminal region of p15. However, this seems unlikely for two reasons. First, the protein without the 6-His tag displayed the same biochemical properties in solution than with the tag, as demonstrated by cross-link experiments. Second, the presence of the His-tag does not modify the structure of the C-terminal region in the truncated form p15-Δ120 compared to the full-length protein. The structure of the C-terminal end appears to be an important feature for retroviral matrices as it differs depending on the virus. Indeed, the C-terminal region following helix h5 is extended in the FIV p15 structure, which is in line with what has been demonstrated for EIAV  and withUnlike SIV, HIV and EIAV MA, FIV p15 appears monomeric in the crystal, although it is dimeric in the protein solution used for crystallization. We could demonstrate that this monomerization of p15 during crystallization was linked to the dissociation of the dimer in the presence of PEG in the crystallization condition. Therefore, it is not clear how the crystallographic contacts we observe might participate to the biological dimer. For example, we demonstrated a shift from a monomer to a dimer in MES buffer at pH 6 but not in phosphate buffer at pH 7.4. This suggests charged residues in the interface of the biological dimer in solution. Unfortunately, the three interfaces that we observed in the crystal involved charged residues that are conserved among FIV strains. It is therefore not possible to discriminate between these interfaces based on their conservation between FIV strains or differences in their protonation susceptibility.in vitro. This suggests that this hydrophobic platform around Trp37 could be involved in the formation of the p15 dimer, which would be destabilized by a PEG molecule that would compete with a p15 monomer for binding to this platform. Previous studies have shown that this Trp contributes to the PIP2 binding site of HIV MA [However, several conclusions can be drawn from the analysis of the three FIV p15 interfaces that were identified in the crystals. In interface 1, Trp37 is implicated in the interaction with C-terminal Pro131 of the complementary monomer  is similar to the buried surface area (410 Å2) of the protein interface leading to the formation of the SIV trimers [A third dimeric interface was identified in FIV p15 crystals, both in the full-length protein and the truncated p15-Δ120. At first glance, interface 3 resembles the interface found in the EIAV dimer , with th trimers .In the absence of native dimers in the crystal structures, due to the dissociation of p15 dimers by the PEG present in the crystallization condition, a specific and systematic mutagenesis study will need to be performed to identify the residues involved in the formation of the dimers. The elucidation of the biological dimeric interface of FIV p15 is important, as dimers of FIV p15 proteins could represent intermediates in the biological multimerization of Gag polyproteins into hexamers. Previous data indicates that although HIV MA proteins form 3D crystals as trimers , no matrIn summary, the dimeric form of FIV p15 in solution and its extended C-terminal end in the crystal structure are original features of this protein when compared to HIV, SIV and EIAV. Although the dimeric interface remains to be identified, the presence of a Trp residue as an interacting platform is likely important for the function of FIV p15. Further study of these characteristics will be needed to better understand their biological relevance in the context of FIV replication and their peculiarity among lentiviral matrix proteins.The FIV p15 open reading frame of the Petaluma strain (GenBank accession number: M25381.1) was amplified from 0.1 μg of the plasmid FIV-34TF10 obtained through the NIH AIDS Research and Reference Reagent Program.CATATGGGGAATGGACAGGGGCGAGATTGG -3', NdeI site underlined), and the reverse primer (5’-AGATCTTCAATGATGATGATGATGATGCTCGAGTGAACCTCTTGG-3') using the Phusion PCR kit (Finnzyme) according to the manufacturer’s protocol, with 25 cycles of PCR and a hybridization temperature of 50°C. A truncated form of the p15 protein (p15-Δ120) was obtained using the same procedure, with reverse primer 5’-GCTCGAGTGAACCTCTTGGAACCAGAGATGGCCTAGTGTCTAATC-3’. A second PCR round was performed on all constructs with the same forward primer and with reverse primer 5'-AGATCTTCAATGATGATGATGATGATGCTCGAGTGAACCTCTTGG-3' introducing the Bgl2 site (underlined) as well as a C-terminal 6-His Tag. The PCR products were digested with NdeI and Bgl2 and inserted into the MCS of the pRSET-B prokaryotic expression vector (Invitrogen) digested with the same enzymes. This creates the pRSET-p15 expression plasmid for our p15 constructs that encode proteins with a 6-His tag at the C-terminal end of the protein. The plasmids were transformed into E. coli BL21(DE3)pLysS cells (Lucigen) for protein expression.For the PCR amplification of the full-length protein, we used the forward primer  to a final concentration of 2 mM. After 20 h of induction at 25°C, cells were collected by centrifugation at 10,000 × g for 8 min, and the wet pellet was stored at -20°C overnight.Cells transformed with pRSET-p15 were grown at 37°C in Hyper Broth (HB) medium (AthenaES™) supplemented with 25 μg/ml of Ampicillin. Transformed cells were grown up in 10 ml of HB for 20 h at 37°C with agitation. This over-grown culture was then added to 1 L of HB supplemented with 25 μg/ml of Ampicillin in a 5 L baffled flask. Cell growth was monitored by optical density (OD) measurements at 600 nm. When culture reached an ODTwo steps of purification were performed to purify FIV p15 proteins: a nickel affinity followed by a size exclusion chromatography.2PO4, 300 mM NaCl), pH 8, with lysozyme (Sigma) to a final concentration of 1 mg/ml, anti-protease cocktail 1X  and DNase I (Sigma) at 2U/ml final, for 1 h on ice. Then, the resuspended pellet was pushed 3 times through a high-pressure chamber, using a microfluidizer M-110P (Microfluidics) at 17,000 psi. The bacterial lysate was clarified by centrifugation at 10,000 × g for 20 min, and the supernatant was filtered through a 0.45 μm membrane. The supernatant was loaded into gravity column packed with 2 g of Ni2+-TED resin (Macherey-Nagel) and eluted with LEW buffer pH 8 containing 50 mM of Imidazole, after 3 washes with LEW buffer pH 8. For gel filtration chromatography, the samples from the affinity chromatography containing the p15 protein loaded on a Vivaspin-PES-5 K filter (Sartorius) and centrifuged at 7,000 × g at 4°C until it reached the volume of 10 ml. The gel filtration experiment was then performed in a FPLC system , using a Superdex 75 TM Hiload 26/60 column  and a buffer containing 50 mM sodium phosphate pH 7.4 or 50 mM MES pH 6, in one injection of 10 ml.A total amount of 10 g of frozen cell pellet from 1 L culture was resuspended in LEW buffer  and centrifuged at 7,000 × g at 4°C until the desired concentration was reached.Purified protein at 1 mg/ml in phosphate buffer was digested overnight at 19°C with 10U of thrombin (Sigma) per mg of protein. After proteolysis, the reaction mix was loaded twice on a Ni-NTA centrifugation column (Proteus) according to the manufacturer’s protocol and the flow-through were collected and pooled. Buffer exchange and protein concentration was then performed as described above for the His-tagged protein.Purified protein in 50 mM phosphate buffer at pH 7.4 or 50 mM MES buffer at pH 6 was concentrated to 4 and 6 mg/ml respectively by using a Vivapsin-PES-5 K centrifugal concentrator (Sartorius). The DLS experiment was realized on a dynamic light scattering Zetasizer Nano-S ZEN1600 instrument  at 20°C. The samples were previously filtered through a 0.02 μm pore size filter . Each measure is the mean of 12 runs and was repeated three times.3, Pierce). Reaction mixtures containing 7 μg of proteins were incubated with 1 μl of 5 mM BS3 (corresponding to a 20:1 (w:w) BS3:p15 ratio) for 30 to 45 minutes at room temperature. The reaction was stopped by addition of 1.5 μl of 250 mM Tris-HCl pH 7.5, 30 mM Glycine for 15 minutes at room temperature, and adjusted to a final volume of 10 μl. Cross-linked proteins were denatured by addition of Laemmli sample buffer and cross-linked proteins were submitted SDS-PAGE analysis. To assess the impact of the crystallization condition, p15 proteins at 6 mg/ml in 50 mM MES buffer pH 6) were mixed to crystallization condition, 0.2 M sodium acetate, or increasing concentration of PEG with a ratio 1:1. Control was performed by mixing the protein with MES buffer only with the same ratio 1:1. Murine antibody 11H6H1 [3 cross-linking in the crystallization condition.Purified p15 proteins at high concentration (6 mg/ml in 50 mM phosphate buffer at pH 7.4 or 6 mg/ml in 50 mM MES buffer at pH 6) and at lower concentration (3 mg/ml in 50 mM phosphate buffer at pH 7.4 or 3.3 mg/ml in 50 mM MES buffer at pH 6) were mixed with a cross-linking agent, the Bis(SulfoSuccinimidyl) Suberate . We loaded 0.2 ml samples of p15 protein at 6 mg/ml in 50 mM MES pH 6, onto a Superdex 200 10/30 gel-filtration column equilibrated at 0.5 ml/min with 50 mM MES pH 6. The eluate was passed successively through a MiniDawn TREOS-angle light scattering detector (Wyatt) coupled to an Optilab T-rEX refractive index monitor (Wyatt). The data were processed, and molecular masses were calculated using the Astra V software (Wyatt).H  and Kd (dissociation constant in mM) were allowed to float during the fit.Dilution experiments were conducted as described previously  to inves3 were obtained within fifteen days. Crystals were transferred to a cryoprotective solution containing the mother liquor and 10% (v:v) ethylene glycol for 30 sec and plunged into liquid nitrogen prior to data collection. First grown crystals diffracted at best to 3 Å resolution at the SLS beamline PXIII  and at the ESRF beamline ID23-2  under cryo-conditions (100 K). Microseeding was necessary to optimize crystals and the best dataset was collected at 100 K to 2 Å resolution at the SOLEIL beamline Proxima 1 . A new crystallization condition was determined for the p15-Δ120 construct. Crystals were obtained in 0.1 M Tris pH 8, 25% (w:v) PEG 6000 (condition 45 of the Qiagen PEGs suite). Crystals were harvested as for the full-length protein and data were collected at 100 K to 2.7 Å resolution at the ESRF beamline ID29.Crystallization conditions were searched using the sitting-drop vapour-diffusion method and commercial kits from Hampton Research, Molecular Dimensions Limited (MDL) and Qiagen. Crystals of FIV p15 were obtained by equilibrating drops of protein (concentrated at 7 mg/ml in 50 mM MES buffer pH 6) mixed 1:1 with the condition 25 of the Qiagen PEGs Suite, consisting of 0.1 M sodium acetate pH 4.6 and 25% (w:v) polyethylene glycol (PEG) 3,000 at 16°C. Tiny rod-shaped crystals with maximum dimension 60 × 30 × 10 μmDiffraction intensities were processed with the programs XDS  and XSCA3 (with a grid spacing of 0.159 Å), large enough to encompass the pocket, was calculated using the AutoGrid program. Lamarckian genetic search algorithm was employed and a maximum of twenty-five million energy evaluations was performed. After docking completion, solutions were ranked and sorted according to their docked energy using the built-in clustering function. Detailed inspection of the ligand-receptor interactions was carried out with AutoDockTools.Molecular docking of the myristoyl group (as ligand) against p15 crystal structure (the receptor) was done using molecular docking program AutoDock 4.2.3 ,65. BothThe sequences alignment between retroviral matrix proteins was carried out using the web tool ESPript/ENDscript ,67. The The coordinates and the structure factors are deposited in the Protein Data Bank  under thThe authors declare no competing interests.JS, PG and CG designed the study. JS, MP, and CG performed the cloning, expression, biophysical characterization and crystallogenesis experiments for both FIV p15 constructs. JS, XR, PG and CG collected, analyzed and interpreted the X-ray diffraction data. XR performed the docking studies. All authors wrote the paper. All authors read and approved the final manuscript.(A) Cross-linking experiment on p15-Δ120 in MES buffer pH 6 at 3 mg/ml (lane 1 & 4) or 6 mg/ml (lane 2 & 5) in the absence (lanes 1–2) or the presence (lanes 4–5) of cross-linking agent BS3. Lane 3: molecular weight marker. (B) Chemical cross-linking with BS3 of control protein FIV p24 [Figure S2: Comparison of the dimeric interface of EIAV (A) with the interface 3 of full-length p15 (B) and p15-Δ120 (C). The color scheme is identical to Figure  FIV p24  in its oClick here for file