Although breast-feeding accounts for 15–20% of mother-to-child transmission (MTCT) of HIV, it is not prohibited in some developing countries because of the higher mortality associated with not breast-feeding. We assessed the potential impact, on HIV infection and infant mortality, of a recommendation for shorter durations of exclusive breast-feeding (EBF) and poor compliance to these recommendations.We developed a deterministic mathematical model using primarily parameters from published studies conducted in Uganda or Kenya and took into account non-compliance resulting in mixed-feeding practices. Outcomes included the number of children HIV-infected and/or dead  at 2 years following each of 6 scenarios of infant-feeding recommendations in children born to HIV-infected women: Exclusive replacement-feeding (ERF) with 100% compliance, EBF for 6 months with 100% compliance, EBF for 4 months with 100% compliance, ERF with 70% compliance, EBF for 6 months with 85% compliance, EBF for 4 months with 85% complianceIn the base model, reducing the duration of EBF from 6 to 4 months reduced HIV infection by 11.8% while increasing mortality by 0.4%. Mixed-feeding in 15% of the infants increased HIV infection and mortality respectively by 2.1% and 0.5% when EBF for 6 months was recommended; and by 1.7% and 0.3% when EBF for 4 months was recommended. In sensitivity analysis, recommending EBF resulted in the least cumulative mortality when the a) mortality in replacement-fed infants was greater than 50 per 1000 person-years, b) rate of infection in exclusively breast-fed infants was less than 2 per 1000 breast-fed infants per week, c) rate of progression from HIV to AIDS was less than 15 per 1000 infected infants per week, or d) mortality due to HIV/AIDS was less than 200 per 1000 infants with HIV/AIDS per year.Recommending shorter durations of breast-feeding in infants born to HIV-infected women in these settings may substantially reduce infant HIV infection but not mortality. When EBF for shorter durations is recommended, lower mortality could be achieved by a simultaneous reduction in the rate of progression from HIV to AIDS and or HIV/AIDS mortality, achievable by the use of HAART in infants. An estimated 2.3 million children under 15 years were living with human immunodeficiency virus (HIV) infection, and 700,000 children were newly infected in 2005 alone [The reduction of HIV transmission during lactation is one of the most pressing global health dilemmas confronting health policy makers and HIV-infected women in many regions of the world -6. ReplaIt has long been recommended that women who are HIV positive should avoid breast-feeding and use replacement-feeding when it is acceptable, feasible, affordable, sustainable and safe (AFASS) . In caseSeveral researchers have modeled the risks and benefits of replacement versus breast-feeding for HIV-infected mothers in developing countries ,7,11-18.a priori to derive parameter sources for this model from Uganda and Kenya, two East African countries where the epidemiology is relatively well documented. In addition, we assessed the impact of variations to the chosen parameters through a sensitivity analysis. In contrast to previous models of time-to-death as a single outcome, we chose to model both cumulative mortality and infection proportions at 2 years. Our choice of these two outcome measures was designed to address the fact that communities may be as concerned with the number of children living with HIV/AIDS after a certain time period as they could be about the number of children dead. Further, cumulative proportions of children living with HIV/AIDS or dead represent statistics that are easy-to-interpret and understand and thus are at least as useful as time-to-event statistics (hazard or rates).Taking these limitations into consideration, we developed a model that examined the potential impact of different infant-feeding recommendations on the overall mortality, burden of HIV and AIDS in children less than 2 years of age, and also examined the impact of varying the duration of breast-feeding and the rate of compliance to infant-feeding recommendations. We chose We developed a compartmental, deterministic model to simulate the effects of different breast-feeding recommendations in HIV-infected women in a typical sub-Saharan setting Figure . This tyB while infants receiving mixed-feeding were infected at a rate of λM. Evidence for higher HIV transmission in infants receiving mixed-feeding when compared to exclusively breast-fed infants[M that were higher than λB. We assumed that pre- and intra-partum use of antiretroviral therapy (ART) by the mother did not have any effect on the cumulative 2-year postpartum risk of HIV transmission through breast-feeding. The base model assumed no use of ART in the postpartum period (as this is not a common practice in many developing countries)[M and λB.The model assumed that post-natal HIV transmission occurred solely through breast-feeding with no difference in the HIV transmission rates by gender. Exclusively breast-fed infants were infected with HIV at a rate of λd infants was takeountries). This asA. We assumed that the mortality in HIV-infected children was mainly due to AIDS-related illness .In addition, we assumed that the risk of HIV transmission in breast milk is constant. By usinB, μF, and μM, respectively.Weaning in exclusively breast-fed infants and infants receiving mixed-feeding occurred at a rate δ; for this simulation, weaning was assumed to be abrupt. This rate was estimated from the inverse of the average duration of breast-feeding in the population. Mortality rates in uninfected infants depended on the mode of feeding with infants receiving exclusively breast-milk, no breast-milk and mixed-feeding dying at rates of μThe following differential equations, with a time step of one week, were used to model the weekly rate at which infants moved in and out of compartments. B(t), F(t), M(t), I(t), and A(t), represented the number of infants respectively in compartments B, F, M, I and A at time t.Six unique infant-feeding scenarios were analyzed Table : these wDt = 104/N, the proportion of children infected with HIV at 2 years defined as (It = 104 + At = 104)/N, and the proportion of children either living with HIV/AIDS or dead at 2 years defined as Dt = 104 + It = 104 + At = 104/N. It is worth noting that the latter combined measure counts each infant only once (not twice). Because this is a compartmental model, at any given time each infant is in one and only one compartment. So at time 2-year, infants who died from HIV will be in the D compartment, not in the I compartment. The assessment endpoint was set a priori at 2 years with the assumption that infant-feeding patterns negligibly affected child mortality after 2 years.The primary model outcomes were: the cumulative mortality at 2 years (104 weeks) defined as Estimates of parameters used in the model were obtained from published articles from a Medline search using the MeSH keywords "Infant + Feeding + HIV" as well as guideline documents published by the World Health Organization publication (WHO). Because of the heterogeneity in parameter values across regions, the parameters were chosen primarily from studies conducted in Kenya or Uganda. For estimates that were not documented in these two countries we used projections from other countries in the region (with similar epidemiology). The specific values used for each parameter are shown in Table Sensitivity analyses were conducted to evaluate the potential impact of the choice of parameters. We performed univariate sensitivity analyses, varying one parameter while holding the rest of the parameters in the model constant. The following parameters were varied: the recommended duration of breast-feeding from 1 to 6 months; compliance to the recommended infant-feeding from 10 to 100%, the proportion of infants born HIV-infected from 1% to 25%; the mortality rates in breast-fed, mixed-fed and replacement-fed infants from 0 to 200 per thousand children, taking into account differences in access to clean water and health care facilities according to geographical settings. Parameters that could be influenced by the greater availability of anti-retroviral therapy such as the rate of infection in breast-fed infants, the rate of progression from HIV to AIDS and the mortality rate due to HIV/AIDS were also varied.In the scenarios with 100% compliance  to the infant-feeding recommendations, exclusive replacement-feeding (scenario U) resulted in the least number of children with HIV/AIDS at 2 years , and 85% compliance when exclusive breast-feeding (for 4 (scenario Z) or 6 months (scenario Y)) was recommended, the number of infants infected with HIV, and having AIDS at 2 years was still lower with replacement-feeding compared to exclusive breast-feeding for 4 or 6 months. However, compared to the scenario where there was 100% compliance, the number of children with HIV/AIDS at 2 years increased by 24% for replacement-feeding with 70% compliance (scenario U vs. X). By contrast, the number of children with HIV/AIDS at 2 years increased only by 1.7% (scenario W vs. Z) and 2.1% (scenario V vs. Y), for infants who were exclusively breast-fed for 4 and 6 months respectively, when compliance was 85% versus 100%. The cumulative mortality increased only by 0.9%, 0.3% and 0.5% in children who had replacement-feeding (scenario U vs. X) or were breast-fed for 4 (scenario W vs. Z) and 6 months (scenario V vs. Y) respectively when compliance was reduced from 100% to 70% for replacement-feeding and 85% for exclusive breast-feeding. When limited compliance was taken into account, the least total number of children with HIV/AIDS or dead at 2 years was obtained when replacement-feeding was recommended (scenario X), followed by exclusive breast-feeding for 4 months (scenario Z) and lastly, exclusive breast-feeding for 6 months(scenario Y)  with increased recommended duration of breast-feeding Figure . By contFor all scenarios, decreasing the compliance to recommended infant-feeding methods resulted in an increase in the number of children infected with HIV and or dead at 2 years Figure . The impVarying the mortality rate in replacement-fed infants did not impact the total number of children with HIV/AIDS at 2 years  in the number of children with HIV/AIDS at 2 years  actually increased mortality, an increase that was accentuated when there was poor compliance. In fact, breast-feeding resulted in the least cumulative mortality only when it was recommended for six months and there was 100% (very high) compliance. Poor compliance could be expected to result in mixed-feeding with its consequences: higher infection rates, more infants who are HIV positive and who die later on. If the aim was solely to reduce mortality, then recommending breast-feeding (including breast-feeding for shorter durations) could only be justified when mortality in replacement-fed infants was greater than 50 per 1000 per year, the rate of infection in exclusively breast-fed infants was less than 2 per 1000 breast-fed infants per week, the rate of progression from HIV to AIDS was less than 15 per 1000 infected infants per week, or the mortality due to HIV/AIDS was less than 200 per 1000 infants with HIV/AIDS per year.Reducing the recommended duration of breast-feeding resulted in fewer children living with HIV/AIDS. However, recommending replacement-feeding (even with poor compliance) resulted in the least number of children living with HIV/AIDS. This suggests that if the aim was solely to reduce the number of children living with HIV/AIDS then replacement-feeding would be the optimal choice irrespective of mortality and rates of HIV transmission and progression.Considering the minimization of the total number of children with HIV/AIDS or dead as main objective, replacement-feeding was the best option in nearly all scenarios, the exception being when the mortality in replacement-fed infants was greater than 150 per 1000 infants or when the mortality rate in replacement-fed infants was more than 3.5 fold that in breast-fed infants.six months unless replacement-feeding is AFASS. It may however be difficult for policy-makers, attempting to implement WHO guidelines, to determine the extent to which replacement-feeding in their settings is AFASS. Our simulations suggest that replacement-feeding may not be considered safe unless the mortality rate in replacement-fed infants is less than three times that in exclusively breast-fed infants.The absence of any reduction in mortality with shorter durations of breast-feeding in this simulation supports WHO's recent update of its guidelines to recommend exclusive breast-feeding for The deterministic nature of this simulation may be a limitation because of its inherent assumption that the parameters and outcomes were fixed (having no variance). Furthermore, as for every mathematical simulation, the conclusions from our analysis could be limited by the veracity of the model selected as well as the limitations of the studies from which parameters were extracted. Our analysis is however strengthened by its particular usefulness to sub-Saharan countries as we used parameters specific to conditions in this high-HIV prevalence setting. Although the parameters used in the baseline model may differ between published studies and may be biased because of defects in the original studies, the impact of varying them was addressed in the sensitivity analysis. This sensitivity analysis of model assumptions showed our findings to be robust within the range of plausible parameters. Furthermore our findings are consistent with a recently published report by Becquet et al. who did not find any significant difference in the 2-year cumulative mortality of exclusively breast-fed infants and replacement-fed infants in West Africa.In conclusion, this analysis presents a framework to assist decision-makers in resource-limited settings in the choice of which infant-feeding method to recommend for infants born to HIV-infected mothers. Recommending exclusive breast-feeding in infants born to HIV-infected women in these settings, instead of replacement-feeding, may potentially result in very little gains in mortality. Furthermore, although recommending shorter durations of breast-feeding may substantially reduce infant HIV infection, it might slightly increase mortality. When exclusive breast-feeding for shorter durations is recommended, lower mortality could be achieved by a simultaneous reduction in of the rate of progression from HIV to AIDS and or HIV/AIDS mortality, reductions that are obtainable by the use of HAART in infants. Making HAART and better care available to infected-infants should thus be an imperative whenever a community and/or policy-maker prefer exclusive breast-feeding over replacement-feeding.AFASS: Acceptable; Feasible, Affordable, Sustainable And Safe, AIDS: Acquired Immune-Deficiency Syndrome; ART: Antiretroviral Therapy; EBF: Exclusive Breast-Feeding; ERF: Exclusive Replacement-Feeding; HAART: Highly Active Antiretroviral Therapy; HIV: Human Immune-deficiency Virus; MTCT: Mother-To-Child Transmission; PCR: Polymerase Chain Reaction; USA: United States of America; WHO: World Health OrganizationThe authors declare that they have no competing interests.JA conceived the study question, designed the study, collected study parameters, conducted the analysis and participated in writing the manuscript. LK designed the study, collected study parameters, conducted the analysis and participated in writing the manuscript. VS designed the study, collected study parameters and reviewed the manuscript. EES designed the study, collected study parameters and participated in writing the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Impact of mortality rate in replacement-fed infants on the Number of Children with HIV/AIDS (top graph), dead (middle graph) or both (bottom graph) at 2 years. U: Exclusive replacement-feeding (ERF) with 100% compliance; V: EBF for 6 months with 100% compliance; W: EBF for 4 months with 100% compliance: X: ERF with 70% compliance; Y: EBF for 6 months with 85% compliance; Z: EBF for 4 months with 85% compliance.Click here for fileImpact of the rate of infection in breast-fed infants on the Number of Children with HIV/AIDS (top), dead (middle) or both (bottom graph) at 2 years. U: Exclusive replacement-feeding (ERF) with 100% compliance; V: EBF for 6 months with 100% compliance; W: EBF for 4 months with 100% compliance: X: ERF with 70% compliance; Y: EBF for 6 months with 85% compliance; Z: EBF for 4 months with 85% compliance.Click here for fileImpact of the rate of progression from HIV to AIDS in infants on the Number of Children with HIV/AIDS (top), dead (middle) or both (bottom graph) at 2 years. U: Exclusive replacement-feeding (ERF) with 100% compliance; V: EBF for 6 months with 100% compliance; W: EBF for 4 months with 100% compliance: X: ERF with 70% compliance; Y: EBF for 6 months with 85% compliance; Z: EBF for 4 months with 85% compliance.Click here for fileImpact of the mortality rate due to HIV/AIDS in infants on the Number of Children with HIV/AIDS (top), dead (middle) or both (bottom graph) at 2 years. U: Exclusive replacement-feeding (ERF) with 100% compliance; V: EBF for 6 months with 100% compliance; W: EBF for 4 months with 100% compliance: X: ERF with 70% compliance; Y: EBF for 6 months with 85% compliance; Z: EBF for 4 months with 85% compliance.Click here for file


The present studies in mice and cancer-bearing patients, treated with C. parvum (CP) immunotherapy, were to determine the effects of CP on the production of immune complexes (IC) and associated disease. Using the Clq-binding assay, circulating immune complexes were detected in mice given a single high dose of CP (466 microgram) and repeated human-equivalent doses (70 microgram). All mice treated with CP developed proliferative glomerulonephritis, the severity of which was dose-related. The histological and immunofluorescent patterns of the nephritis were those attributed to immune-complex disease. The mice had haematuria but were not in renal failure. Fifty patients with inoperable lung cancer were studied. All received radiotherapy. Twenty-two had no other treatment (controls) and 28 were treated with infusions of CP. Using 2 immune-complex assays  IC were found in 10/22 control patients but these did not develop haematuria or proteinuria. Twenty-four of the 28 patients treated with CP developed transient haematuria and/or proteinuria with red-cell and hyaline casts, the changes resolving over 5 days. Immune complexes were detected in 5 of these 28 patients before CP treatment. Although 16/28 had IC at the time of haematuria and proteinuria, these findings were difficult to interpret because IC may occur in response to the tumour, the radiotherapy, or the CP. Although no patient developed renal failure, we believe that those treated with CP should have regular assessment of their renal function.


Escherichia coli and Klebsiella species (ESBL-EK) have not been adequately investigated.Clinical outcomes of spontaneous bacterial peritonitis (SBP) due to extended-spectrum β-lactamase-producing We conducted a retrospective matched case-control study to evaluate the outcomes of SBP due to ESBL-EK compared with those due to non-ESBL-EK. Cases were defined as patients with liver cirrhosis and SBP due to ESBL-EK isolated from ascites. Control patients with liver cirrhosis and SBP due to non-ESBL-EK were matched in a 3:1 ratio to cases according to the following five variables: age (± 5 years); gender; species of infecting organism; Child-Pugh score (± 2); Acute Physiological and Chronic Health Evaluation II score (± 2). 'Effective initial therapy' was defined as less than 72 hours elapsing between the time of obtaining a sample for culture and the start of treatment with an antimicrobial agent to which the EK was susceptible. Cephalosporin use for ESBL-EK was considered 'ineffective', irrespective of the minimum inhibitory concentration. ESBL production was determined according to the Clinical and Laboratory Standards Institute guidelines on stored isolates.vs. 10/78, 13%, P = .006); 30-day mortality rate was also higher than in the controls . When the case were classified according to the effectiveness of the initial therapy, 'ineffective initial therapy' was associated with higher 30-day mortality rate .Of 1026 episodes of SBP in 958 patients from Jan 2000 through Dec 2006, 368 (35.9%) episodes in 346 patients were caused by SBP due to EK, isolated from ascites. Of these 346 patients, twenty-six (7.5%) patients with SBP due to ESBL-EK were compared with 78 matched controls. Treatment failure, evaluated at 72 hours after initial antimicrobial therapy, was greater among the cases (15/26, 58% SBP due to ESBL-EK had poorer outcomes than SBP due to non-ESBL-EK. Ineffective initial therapy seems to be responsible for the higher rate of treatment failure and mortality in SBP due to ESBL-EK. Escherichia coli and Klebsiella species (EK) are the most common cause of SBP [rd generation cephalosporins, such as cefotaxime, have been viewed as the drug of choice for empirical treatment of SBP [Spontaneous bacterial peritonitis (SBP) is a major cause of morbidity and mortality in cirrhosis patients . Gram nee of SBP ,3. Histoe of SBP -6. 3rd gt of SBP .Escherichia coli and Klebsiella species (ESBL-EK) has increased. Several studies of clinical outcomes in patients with infections due to ESBL-producing organisms have shown higher mortality and reduced rates of clinical and microbiologic response compared to infections due to non-ESBL-producing organisms [Since the 1980s, the incidence of infections due to extended-spectrum β-lactamase-producing rganisms . HoweverTo identify patients with advanced liver cirrhosis and SBP due to EK isolated from ascites, we reviewed the database at the clinical microbiology laboratory, and the medical records, by the diagnosis at discharge from 1 Jan 2000 to 31 Dec 2006. EK which was not susceptible to either cefotaxime or ceftazidime was considered 'suspected ESBL-EK'. This study was approved by the institutional review board by Seoul National University Hospital.Most of the ascitic isolates were collected by the clinical microbiology laboratory in our hospital. Species identification was carried out by standard methods with VITEK-GNI cards .E. coli ATCC 25922 and K. pneumoniae ATCC 700603, were inoculated in each set of tests for quality control.The antibiotic susceptibility of each isolate was determined by the disk diffusion method, using the criteria of the Clinical and Laboratory Standards Institute (CLSI). ESBL production was determined by the disk diffusion method according to CLSI performance standards. Briefly, we determined the diameters of the inhibition zones on cefotaxime and ceftazidime disks (30 μg each), alone and in combination with clavulanic acid (10 μg). An increase of ≥5 mm in zone diameter when either of the antimicrobial agents was combined with clavulanic acid was considered evidence of ESBL production. Two control organisms, Cases were defined as patients with advanced liver cirrhosis and SBP due to ESBL-EK, isolated from ascites. Controls were patients with advanced liver cirrhosis and SBP due to non-ESBL-EK. They were matched in a 3:1 ratio to case patients according to the following five variables: age (± 5 years); gender; species of infecting organism; Child-Pugh score (± 2) ; Acute PWe reviewed the medical records of both case and control-patients. The data collected included: age, gender, species of infecting organism, Child-Pugh score, severity of illness calculated by the APACHE II score, presentation with septic shock, presence of bacteremia, care in the intensive care unit (ICU) and antimicrobial regimen. The following conditions were also documented to identify 'risk factors' for infection by ESBL-producing microorganism: hospital stay before onset of SBP, presence of hepatocellular carcinoma, history of SBP, antimicrobial therapy within the 30 days prior to onset of SBP, neutropenia, presence of central venous catheter, indwelling urinary catheter, use of immunosuppressive agents including corticosteroid within 30 days, polymicrobial infection and invasive procedure within 72 hours prior to SBP.Primary outcomes were the initial treatment response, and 7-day and 30-day mortality rates. The initial treatment response was assessed 72 hours after starting antimicrobial therapy and was classified as follows: 'complete response' for patients who had resolution of fever, leukocytosis and all signs of infection; 'partial response' for patients who had abatement of abnormalities in the above parameters without complete resolution; 'failure' for patients who had died or deterioration or absence of abatement of fever, leukocytosis and all other signs of infection .The diagnosis of SBP was based on the combination of a positive ascitic fluid culture and a polymorphonuclear leukocyte (PMNL) count of >250 cells/μL, or on a combination of symptoms/signs of SBP and a PMNL count of >250 cells/μL . Neutrop'Effective initial therapy' was defined as less than 72 hours elapsing between the time of obtaining a sample for culture and initiation of treatment with an 'effective' antimicrobial agent. The antimicrobial therapy was considered 'effective' if the treatment regimen included antibiotics active in vitro, and the dosage and route of administration were in conformity with current medical standards. Cephalosporin monotherapy for ESBL-EK was considered 'ineffective', irrespective of the minimum inhibitory concentration.P values were two-tailed, with P < 0.05 being considered statistically significant. We performed univariate analysis for the relationship between risk factors and infection by ESBL-producing EK using conditional logistic regression. SPSS software version 15.0  was used for these analyses.The Pearson chi-square test, Fisher's exact test, Wilcoxon signed rank test and Kaplan-Meier survival analysis were used to compare clinical variables and outcomes, as appropriate. All From 1 Jan 2000 to 31 Dec 2006, 1026 episodes of SBP were identified in 958 patients. Of the 1026 episodes, 368 (35.9%) in 346 patients were diagnosed as SBP due to EK isolated from ascites. Of the 368 episodes, 32 (8.7%) were due to 'suspected ESBL-EK'. All 32 strains of 'suspected ESBL-EK' were stored, and all but one were recovered. Each 'suspected ESBL-EK' caused one episode of SBP in one patient. We performed a confirmatory test for ESBL-production on these 31 EKs. Twenty-six were confirmed as producing ESBL. These 26 patients were included in the case group and the triple number of patients was selected for the control group. Clinical characteristics including five matched-variables in these patients are shown in Table vs. 13%, P = .006). The 30-day mortality rate of the cases was also higher than that of the controls .Clinical outcomes and survival curves for SBP are shown in Table rd generation cephalosporin. Of the 18 case patients who had ineffective initial therapy, two died within 72 hours of the start of the empirical treatment. Apart for these two patients, the remaining 16 patients received antibiotics (carbapenems) effective against ESBL-EK after receipt of reports of the susceptibility tests. The clinical characteristics and outcomes of the patients with SBP due to ESBL-EK are shown in Table vs. 61%, P = .036).Of the 26 case patients, eight 31%) had effective initial therapy with imipenem, whereas all of the control patients received effective initial therapy with 31% had efThe risk factors associated with SBP due to ESBL-EK are listed in Table In a previous study , we demoIn patients with liver cirrhosis, Child-Pugh and APACHE II scores are known to be the most important factors influencing mortality due to SBP . We trieet al. and our previous work showed that a delay in effective treatment for ESBL-producing organisms did not result in poorer clinical outcomes in patients with urinary tract and bloodstream infections generally originating from the pancreatobiliary tract [et al. demonstrated that inadequate initial therapy was an independent risk factor for mortality in ESBL-EK infections [Among the other factors influencing the outcomes of ESBL infection, the impact of a delay in effective initial treatment was controversial. Lautenbach ry tract ,18. In cfections . In the Therefore, it would seem reasonable to treat patients with SBP due to ESBL-EK with effective antibiotics straight away. However, SBP due to ESBL-EK accounted for only 7.5% (26/346) of the SBP due to EK, and the injudicious use of broad antimicrobial regimens is likely to result in further emergence of resistance. We suggest that one should use antibiotics active against ESBL-producing organisms in those selected patients who have a high risk of infection. The risk factors for the development of infection with ESBL-producing organisms that have been listed in previous studies are: length of hospital stay and ICU stay, presence of central venous catheter, prior administration of an antibiotic, and severity of illness etc ,18,20,21Our study has potential limitations. First, we did not evaluate the 'attributable' mortality of SBP; some deaths in our study may not have been related to the SBP. However, efforts to designate outcomes as 'attributable' to infection are often subjective and inconsistent ,23. We tOur results indicated that SBP due to ESBL-EK had poorer outcomes than SBP due to non-ESBL-EK and 'ineffective initial therapy' may be a cause of the higher rates of treatment failure and mortality in SBP due to ESBL-EK. However, SBP due to ESBL-EK accounted for only 7.5% (26/346) of the SBP due to EK, and the imprudent use of broad spectrum antibiotics should be avoided. Therefore, studies aimed at identifying the risk factors for SBP caused by ESBL-producing organism are needed in order to optimize initial therapy for selected patients who have a high risk of infection.The authors declare that they have no competing interests.All authors conceived of the study. KHS and JHJ collected the data. KHS and NJK carried out data analysis and interpretation. WBP, SWP, HBK, MDO, HSL and KWC carried out data interpretation. KHS and NJK drafted the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/41/prepub


Micro(mi)RNAs regulate gene expression through translational attenuation and messenger (m)RNA degradation, and are associated with differentiation, homeostasis and disease. Natural miRNA target recognition is determined primarily by perfect complementarity in a seed region (nucleotide positions 2 to 7) with additional interactions contributing in a sequence- and target-specific manner. Synthetic miRNA target analogs, which are fully complementary, chemically modified oligonucleotides, have been used successfully to inhibit miRNA function.In this paper, we present a first systematic study to evaluate the effect of mismatches in the target site on synthetic inhibitor activity. Panels of miRNA inhibitors containing two-nucleotide mismatches across the target site were tested against three miRNAs . The results showed that the function of inhibitors vary as mismatch positions in the inhibitors change.The data indicate that features important for natural miRNA target recognition (such as seed region complementarity) are also important for inhibitor functionality. In addition, base pairing at a second, more 3' region appears to be equally important in determining the efficacy of synthetic inhibitors. Considering the importance of these inhibitor regions and the expression of closely related miRNA sequences will enable researchers to interpret results more accurately in future experiments. Micro (mi)RNAs are small (17 to 27 nucleotides), non-coding RNAs that act in association with Argonaute (Ago) proteins to modulate gene expression via an effector nucleic acid-protein complex (microribonucleoprotein (RNP) or miRNA-induced silencing complex (RISC)). In animals, miRNA-based gene modulation occurs predominantly by the mature miRNA binding to an mRNA target site through partial base pairing, resulting in translational attenuation . ComputThe functional roles of miRNAs can be investigated using inhibitors, which are nucleic acid-based molecules that suppress miRNA function. Synthetic miRNA inhibitor designs incorporate the reverse complement of the mature miRNA (the target site) and are chemically modified to prevent RISC-induced cleavage, enhance binding affinity and provide resistance to nucleolytic degradation (for review see ). When dTo correctly associate outcomes of inhibitor experiments with specific miRNAs, it is important to understand the degree to which an inhibitor designed against one miRNA affects other miRNAs. We used synthetic inhibitors and luciferase reporters targeted by individual miRNAs to study inhibitor specificity among both natural miRNA variants in a multi-member family (let-7) and artificially designed inhibitor variants to single miRNAs . Strong inhibitor crossreactivity between members of the human let-7 family, which share extensive sequence identity, was observed. Inhibitors to three different human miRNAs  were systematically mismatched at all positions, and two regions that affect inhibitor specificity were identified: the seed region (positions 3 to 8) and an additional 3' region (positions 13 to 18). These results will aid in interpretation of synthetic miRNA inhibitor studies and improvement of experimental design.To gain insight into the level of inhibitor crossreactivity to be expected between closely related family members, hairpin inhibitors (see Methods) designed against each of the nine human let-7 miRNAs Figure  were choTo determine crossreactivity between unrelated (non-family member) miRNA inhibitors, we focused our studies on miR-21 and miR-122. Both miRNAs are the sole human representatives of their respective families, but were found to have about 52% sequence similarity Figure . To testIn the studies described above, we found that inhibitors for miRNAs that differ at only a few nucleotides crossreacted, whereas miRNAs that differ at multiple 7 to 10) nucleotides did not. To study the effect of mismatches in inhibitor target sites (the site where the miRNA binds) on inhibitor functionality, systematically mismatched inhibitors were tested for miRNAs represented by only a single family member (for ease of data interpretation) in cell lines in which they are strongly expressed   and a second region closer to the 3' end (positions 13 to 18) Figure . The purIn this study, it was observed that changes in inhibitor efficacy due to mismatches were affected by miRNA expression levels and inhibitor concentration. Endogenous miRNA downregulation of the luciferase reporter, compared with the psiCHECK-2 vector without insert, suggests that miR-21, miR-22 and miR-122 are expressed at varying levels . The differences observed for position 13 relative to function of the inhibitors are probably due to the nature of the modification pattern used (two mismatches versus one mismatch) or to the sensitivity of the assay system employed. That is, subtle changes are more readily observed in cell culture with a synthetic reporter than in a mouse model documenting steady-state levels of miR-122 (by northern blotting) and target gene mRNA (by reverse transcriptase PCR) in liver preparations.Although few systematic studies of miRNA inhibitor specificity have been reported in the literature, the results from the current study are similar to those from a recent report of synthetic inhibitors in an t et al.  tested fO-methyl nucleotide modifications increase binding affinity to the mature miRNA and this limits comparison of our study with the miRNA/siRNA-mRNA studies.Although regions of importance common to all three miRNA inhibitors tested were very evident, there were slight differences observed in inhibitor responses to mismatches at particular locations. For example, within the seed region, the 3 to 4 mismatch was especially detrimental for miR-21 inhibitor function, whereas it was the least perturbing for miR-22 inhibitor function. One reasonable explanation for such differences may be sequence dependence, but in the example given, the mismatched pair was CG for both miR-21 and miR-22  that produces irreversible sequestration of the miRNA and the endogenous miRNA-mRNA (RNA-RNA) interaction that results in gene modulation.In our study, we found positions 3 to 8 in the inhibitor to be important for miRNA inhibition. These data are in agreement with many other studies, which have found the seed region to be a determinant of miRNA-mRNA target recognition . TherefoThe 3' region, positions 13 to 18, found in the current study to be crucial for miRNA-inhibitor recognition, roughly corresponds to the '3' compensatory' or 'beneficial 3' pairing' sites identified in miRNA-mRNA recognition of target sites in expressed reporter 3' untranslated regions ,12. Howein vitro studies of miRNA duplex unwinding in Drosophila melanogaster . Mismatched inhibitors contain two consecutive mismatches  across the target site. Sequences for all inhibitor target sites in this study are reported in Figure All inhibitors were fully 2'-CO, USA)  , was cultured in modified Eagle's medium (high glucose) with 10% fetal bovine serum and 2 mM L-glutamine. This cell line is known to have moderate to high endogenous levels of miR-122 ,25,37. A® Duo [Thermo Fisher Scientific] or cat. no. 11668; Lipofectamine 2000 ) according to the manufacturers' instructions, at 0.2 to 0.4 μg/well. Inhibitor concentrations varied between approximately 0.03 and 21 nM, whereas reporter and control plasmid concentrations remained constant at 100 ng/well.For transfection, cells were plated into 96-well plates, with 10,000 cells/well in serum-containing media without antibiotics, and transfected approximately 24 hours after plating. For luciferase assays, the test inhibitor molecule and the respective dual luciferase reporter or control plasmid  were co-transfected into cells using transfection reagents  gene (NM_000034) response to miRNA modulation, cells were transfected with the miR-122 hairpin inhibitor over a concentration range of 0.8 to 100 nM using 0.2 μg/well transfection reagent , according to the manufacturer's instructions. In cases where cells were cultured for longer than 3 days post-transfection, the media was replaced with new growth media approximately every 3 days.http://www.mirbase.org/) cloned into the the 3' untranslated region of the modified Renilla luciferase (hRluc) gene. These constructs were used in conjunction with a commercial assay system . Because this assay measures protein activity, these reporters show luciferase response from both cleavage and non-cleavage targeting [et al. [Renilla luciferase/firefly luciferase (Rluc/Fluc) values are the average of triplicate wells; error bars are ± 1 SD (sample) of the original average, scaled for subsequent ratio calculations. Reported values are the ratio of the reporter plasmid signal to the control plasmid (psiCHECK-2) signal normalized to the ratio obtained from co-transfection with matched amounts of non-targeting inhibitor controls.The dual-luciferase reporters were all derived from the psiCHECK-2 vector  and contain a single target site, fully complementary to the mature miRNA -40  according to the manufacturer's instructions. For all data reported in this study, no cell viability differences were observed between the reporter and control (psiCHECK-2) plasmid treatments (data not shown).Cell viability was assessed using an indicator dye   mRNAs were determined using the branched DNA assay  [Levels of both ALDOA and peptidyl-prolyl CA, USA) . KnockdoThe authors declare that they have no competing interests.BR conceived of and designed the specificity studies, carried out assays and analyzed results, and drafted the manuscript. AD carried out luciferase assays and analyzed the results. AK participated in design and in interpretation of results. JK participated in the design, analysis of results and drafting, and critical review of the manuscript. DL participated in the design, analysis of results and critical review of the manuscript. AV participated in design, coordination of execution and analysis of all experiments, and drafting and critical review of the manuscript. All authors read and approved the final manuscript.Supplemental Figures and Tables. Supplemental Figure S1 - All let-7 family member reporter assays show crossreactivity between all let-7 inhibitors, although the degree of apparent crossreactivity varies across reporters. Supplemental Figure S2 - Dose curves of fully complementary inhibitors co-transfected with cognate reporters. Supplemental Table S1 - Sequences of let-7 inhibitor target sites. Supplemental Table S2 - Sequences of mismatched inhibitor target sites.Click here for file


A primary aim of the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort study is to examine the association between total physical activity levels  and the incidence of cancer. We examined the validity and long-term repeatability of total physical activity measurements estimated from the past-year recall EPIC questionnaire, using accelerometers as an objective reference measure.Participants included 100 men and 82 women aged 50–65 years. Criterion validity was assessed by comparing the physical activity estimates from the EPIC questionnaire with total activity estimated from the average of three separate 7-day accelerometer periods during the same (past-year) period. Long-term repeatability of the EPIC questionnaire was assessed by comparing the responses from the baseline and 10-month administrations. Past-year EPIC estimates were also compared with the Friedenreich Lifetime Total Physical Activity Questionnaire to examine whether recent activity reflected lifetime activity.Accelerometer total metabolic equivalent (MET)-hours/week were positively associated with a total physical activity index  0.15, 0.42) and with non-occupational activity estimated in MET-hours/week . Stratified analyses suggested stronger correlations for non-occupational activity for participants who were male, had a lower BMI, were younger, or were not full-time workers, although the differences in correlations between groups were not statistically significant. The weighted kappa coefficient for repeatability of the total physical activity index was 0.62 . Spearman correlations for repeatability of components of activity were 0.65  for total non-occupational, 0.58  for recreational and 0.73  for household activity. When past-year activity was compared to lifetime estimates of activity, there was fair agreement for non-occupational (ρ = 0.26) activity, which was greater for household activity (ρ = 0.46) than for recreational activity (ρ = 0.21).Our findings suggest that the EPIC questionnaire has acceptable measurement characteristics for ranking participants according to their level of total physical activity. The questionnaire should be able to identify the presence or absence of reasonably strong aetiological associations when either recent or long-term activity is the responsible factor. Physical activity is an important modifiable risk factor for several types of cancer, including colon and breast cancers, and possibly prostate, endometrial and lung cancers -4. For pThe European Prospective Investigation into Cancer and Nutrition (EPIC) on-going prospective cohort study was initiated primarily to examine the associations between diet, lifestyle and the incidence of cancer in over 500,000 participants in 10 western European countries. The EPIC questionnaire assesses past-year physical activity in occupational, recreational and household domains ; its valAs the EPIC study will continue to provide important results on the association of physical activity with risk of developing cancer and other chronic diseases, and these results will be incorporated into population-level physical activity guidelines, data from our validation study will enable better interpretation of EPIC study findings on total physical activity and cancer risk, and give guidance for use or adaptation of this questionnaire in other studies.The objectives of this study were 1) to assess the criterion validity of total physical activity estimated from the EPIC questionnaire, using three 7-day accelerometer periods as an objective reference measure; 2) to evaluate the long-term repeatability of the EPIC questionnaire over 10 months; and 3) to compare agreement between the EPIC questionnaire and the Friedenreich Lifetime Total Physical Activity Questionnaire (LTPAQ) .Eligible study participants included men and women aged 50–65 years living in Sydney, New South Wales (NSW), Australia. Volunteer participants were recruited between June and November 2005, from NSW state-wide Health Survey participants (63% of cohort), workplaces (33%), and by word-of-mouth (4%). Compared to the NSW general population, our study had a slightly higher proportion of participants who were male, or employed . In our Eligible participants were contacted initially by mail, with follow-up by mail and telephone. Of 401 people who were sent an information package, 189 (47%) gave consent and completed baseline data collection and 186 completed all aspects of the study including three accelerometer monitoring periods and the final questionnaire. After exclusion of four subjects with insufficient accelerometer data, 182 participants were included in the analysis.Each participant completed the EPIC self-administered and LTPAQ telephone-administered physical activity questionnaires at baseline. During follow-up, subjects wore an Actigraph (MTI) accelerometer for three separate 7-day periods, each 14 weeks apart to capture seasonal variation. The EPIC questionnaire was completed again about 10 months after baseline  to create a total physical activity index categorised as inactive, moderately inactive, moderately active, and active [see Additional file The Friedenreich LTPAQ records at interview the recalled frequency , duration  and intensity of each of a respondent's physical activities in four different domains  over the entire lifetime . It has Data were processed according to the current LTPAQ protocol (available from C.M.F.). MET values  were assParticipants wore an Actigraph accelerometer  ,21 on thThe monitor was initialized as described by the manufacturer and data collected in 1-minute epochs. Activity counts above 18,000 counts/min were censored as probable artefact. Consecutive strings of zero-count epochs lasting 20 minutes or more were assumed to be periods of non-wear . The aveThe Swartz prediction equation and cut-points  were useTotal physical activity was the main measure of interest, measured primarily using the 'total physical activity' categorical index, and secondarily as non-occupational activity in MET-hours/week from the EPIC questionnaire. Criterion validity was assessed by comparing these physical activity measures, as estimated from the EPIC questionnaire at 10 months, with total activity  estimated from the average of three 7-day accelerometer measures. The long-term repeatability of the EPIC questionnaire was assessed by comparing total activity, and activity in different domains  and intensities , between the baseline and 10-month administration. Recent and lifetime physical activity were compared using the baseline EPIC and LTPAQ questionnaires.As the data were not normally distributed, we used nonparametric tests and presented data as medians and interquartile ranges. Spearman correlation coefficients (ρ) with 95% confidence intervals  and Bland-Altman plots with 95% limits of agreement  were cal2 (range 18.6–61.7) and 70% of participants were classified as overweight or obese. More than half the participants were in full-time employment .A total of 100 men and 82 women with a mean age of 57.2 years participated in this study. Baseline characteristics of participants are shown in table Pdiff <0.0001). The estimated hours/week of non-occupational activity was 20.3 using the EPIC questionnaire compared with 12.2 for the LTPAQ (Pdiff <0.0001). Full-time workers and non-workers/casual-workers had similar estimates of total activity using accelerometers, but when the EPIC non-occupational activity estimates were stratified by employment status, the median hours/week were 17.1 for full-time workers and 29.2 for non-workers/casual-workers (Pdiff <0.0001), which gives an indication of the overall contribution of occupational activity to total activity in this sample. Light to moderate intensity activities were the most prevalent activities in our population, accounting for more than 99% of total activity based on the accelerometer data.Physical activity levels as estimated by the different assessment methods are reported in table Box-and-whisker plots figure  demonstrPdiff >0.10), and we had limited power to detect these differences (<30% power for most inter-method subgroup comparisons in this study). The correlation for vigorous-intensity activity was 0.23 when MET-assigned intensities were used, and 0.18 when using self-rated intensity (table Table P = 0.17). The correlation for non-occupational activity was slightly higher  when the average of both EPIC questionnaires was compared to the LTPAQ.The correlation for the agreement of MET-hours/week between the baseline EPIC and LTPAQ questionnaires was 0.26 for total non-occupational activity, 0.21 for recreational activity, 0.46 for household activity, 0.40 for vigorous activity and 0.26 for light-moderate activities (table Pdiff = 0.01). Light-moderate activities and vigorous activities had similar test-retest correlations. The weighted kappa coefficients were similar for the 'total' and 'Cambridge' index . Eighty-four percent of participants reported the same level of occupational activity on both administrations. The test-retest correlations were generally similar according to sex, BMI, age and employment status, although for household activity the correlation was higher for older  than younger  participants (Pdiff <0.001). Although the mean difference in MET-hours/week of non-occupational activity was small, a Bland-Altman plot of these data (not presented) showed wide 95% limits of agreement . A similar pattern was seen for recreational activity, and to a lesser extent, household activity (data not shown). When individual activities were examined, the repeatability was highest for housework (ρ = 0.77), followed by gardening (ρ = 0.73), sports and stair-climbing (both ρ = 0.69), cycling (ρ = 0.65), do-it-yourself activity (ρ = 0.47) and walking (ρ = 0.41).The repeatability of the EPIC physical activity questionnaire over 10 months is shown in table The primary consideration of physical activity assessment in cancer epidemiological studies is to ensure appropriate and consistent categorisation of participants according to their total physical activity level. Our findings suggest fair agreement between the EPIC questionnaire and accelerometer measurements in the ranking of physical activity level, and satisfactory long-term repeatability of the EPIC questionnaire over a 10-month interval.Estimates of total physical activity, encompassing occupational, recreational and household activity from the EPIC questionnaire, are based on a four-level 'total physical activity index'. This index was positively correlated ρ = 0.29) with increasing accelerometer measurements, suggesting that the index is suitable for ranking overall level of total physical activity. However, in our study, the index appeared better at distinguishing the most active participants than those in the lower activity categories. The more active participants may have clearly defined patterns of physical activity that are more easily recalled, which could partly explain the greater agreement. There were similar positive associations for both the Cambridge index (ρ = 0.32) and the occupational level classification ρ = 0.37). For each of these three indices there was a clear distinction between the most active and the least active group. Previously, studies have used heart rate monitoring [ with inc. For eacHowever, although the EPIC instrument can suitably rank participants according to physical activity level, there remains a considerable level of measurement error when assessing an individual's physical activity. Physical activity questionnaires with similar measurement characteristics to the EPIC questionnaire have been shown to lead to substantial attenuation of relative risk estimates for associations between physical activity and an outcome of interest, assuming the measurement errors are non-differential . For exaThe correlation between non-occupational physical activity from the EPIC questionnaire and accelerometer measurement was 0.21 overall, suggesting weak to fair agreement. It may, however, underestimate the true level of agreement because the accelerometer measurements include occupational physical activity, which is not necessarily well correlated with non-occupational activity since people who are physically sedentary at work might compensate by doing more recreational activity or vice-versa. In this population, for example, the correlation between these two components from the LTPAQ questionnaire was -0.12 . Thus, non-occupational physical activity is likely to be a better measure of total activity among those who are not employed, which is supported by our stratified results . Despite this probable underestimation, the correlation of EPIC with accelerometer measurements is within the range of validity coefficients that have been shown with other self-report measures of adults' habitual or global physical activity: generally 0.14 to 0.36 ,30,31. Aet al [et al [The weighted kappa coefficients for the repeatability of the total physical activity index (0.62) and the Cambridge index (0.66) indicate good agreement  in classet al  reportedet al ,31. Usinl [et al  observedMeasures of total and vigorous physical activity generally have higher repeatability coefficients than light-moderate intensity activities because they are usually more easily recalled ,33-35. HPdiff = 0.008), which may reflect the more variable nature of recreational activities throughout life compared to household activities that are regularly performed [Long-term exposures are thought to be more important than recent exposures in the aetiology of most cancers. Our data comparing the EPIC and Friedenreich LTPAQ questionnaires suggest that recent physical activity partially reflects lifetime activity, as recalled by the participants. The correlations were significantly higher for household activity than for recreational activity , and they are limited in their ability to monitor upper body movements, water activities and movements with a weak vertical component such as cycling ,42,43. TFor future use in epidemiological studies, some minor changes could be incorporated into the EPIC questionnaire that may improve its measurement characteristics and distinguish better between people who are sedentary or moderately inactive. Suggested improvements include i) capturing the frequency and duration of occupational activity, ii) changing the question on vigorous activity to mention 'breathing much harder than normal' rather than focus on 'sweating' (which can be weather dependent), and iii) splitting the housework activities into two or more categories , to assist with recall and to allow more precise estimation of intensity levels.Our findings suggest that the EPIC questionnaire has acceptable measurement characteristics for ranking participants according to their level of total physical activity. The EPIC questionnaire should be able to identify the presence or absence of reasonably strong aetiological associations when either recent or long-term activity is the responsible factor.EPIC: European Prospective Investigation into Cancer and Nutrition; BMI: body mass index; CI: confidence interval; MET: metabolic equivalent values; ρ: Spearman rank correlation coefficient; SD: standard deviation; LTPAQ: Lifetime Physical Activity Questionnaire.The authors declare that they have no competing interests.AEC was the primary author responsible for design, statistical analysis, interpretation of data, and drafting and revising the manuscript, BJS, BKA, and AB helped conceive the study and formulate the study design, BJS, JC, HPV, CMF, BKA, and AB contributed to the interpretation of the data and critically revised the manuscript for important intellectual content, AEC, BJS, and JC contributed to collection of data. All authors read and approved the manuscript.The EPIC physical activity questionnaire.Click here for fileThe total physical activity index, based on the cross-classification of occupational activity with combined recreational and household activity.Click here for fileThe classification of physical activity according to the Cambridge Physical Activity Index.Click here for file


Phosphatidylethanolamine (PE) is a phospholipid which is biosynthesized into long chain N-acylethanolamines (NAEs) including oleoylethanolamide (OEA), a known inhibitor of food intake. The aim of this study was to investigate whether PE-containing lipids can also inhibit intake. This was a 4 treatment intervention where 18 male participants were given a high-fat test breakfast  containing (i) high-phospholipid, high-PE lipid (ii) high-phospholipid, medium-PE lipid (iii) no-phospholipid, no-PE control lipid or (iv) water control, in a randomised cross-over. Visual analogue scales (VAS) were used to assess post-ingestive hunger and satiety, and energy intake (EI) was measured at an ad libitum lunch meal after 3.5hours.When compared with the water control, the 3 lipid treatments resulted in lower levels of hunger and thoughts of food, greater fullness and satisfaction , and a lower EI (P<0.05). However, there was no difference in any of the VAS measures when the 2 PE lipid treatments were compared with no-PE control lipid, nor when medium-PE was compared with high-PE. Unexpectedly participants ate significantly more energy at the lunch meal when the 2 PE lipid treatments  were compared with the no-PE control lipid , although there was no dose effect between the medium- and high-PE treatments.Despite the close relationship of PE with OEA, there was no evidence from this acute study that dietary phospholipids containing PE can favourably modify eating behaviour. The inability to control energy intake (EI), compounded by low levels of physical activity, appears key to the development of obesity. Regulation of appetite is central to the control of weight gain, and identifying foods which can modulate hunger and satiety and hence decrease intake is an area of considerable interest. There is a body of data which suggests that dietary lipids may be the least satiating of the 3 food macronutrients -4, but tN-acylethanolamines (NAEs), and which include N-arachidonoylethanolamine (anandamide) and its analogue oleoylethanolamide (OEA) [Phospholipids (PL) are crucial lipid components of biological membranes and, whilst present in the diet in much smaller gram amounts than triglycerides, are found typically in items including eggs, dairy, liver and oils such as soybean. Whilst phosphatidylcholine  is the most abundant PL in animal tissues , phosphade (OEA) . OEA is de (OEA) ,16. OEA de (OEA) ,17 and ode (OEA)  into londe (OEA)  and mainIn light of the novel anorectic effects of OEA, this trial was designed to investigate whether dietary PE may also enhance satiety. To determine whether PE-containing PLs affect appetite regulation, a high-fat test-breakfast containing zero, medium or high doses of PE were given to a group of healthy male participants. Hunger ratings were measured through the morning and EI covertly measured at a buffet style meal presented for lunch.Eighteen lean, healthy men completed this 4 treatment trial. Baseline characteristics are provided in Table post-hoc analyses were carried out, there was no significant difference between the 3 lipid-containing treatments in pleasantness, visual appeal, smell, taste, after-taste and palatability (P > 0.05) demonstrating that perceived differences in the test-breakfast were not responsible for any differences in EI that may later occur at the ad libitum lunch. There was also no significant difference when the 2 PE treatments were compared with the no-PE control lipid (P > 0.05).The lipid containing test-breakfasts  were designed to be as similar in appearance and taste as possible, with no significant difference in total energy, fat, carbohydrate (CHO) or protein content, as shown in Table ad lib lunch) both hunger and fullness had gradually returned to close to fasting levels. There was no change in VAS measures for the water control. As expected, when analyzed over the 3.5 h post-test-breakfast period, there was a significant effect of the 4 treatments on hunger, fullness, satisfaction and thoughts of food , again a consequence of inclusion of the no-energy water control in the analyses. There was also a significant difference between the treatments over time . When post-hoc analyses were carried out and the two PE treatments combined and compared with water control, there was a significant difference for all 4 VAS measures between treatments , but there was no significant difference in hunger, fullness, satisfaction or thoughts of food when compared with the no-PE control lipid (P > 0.05). When the high-PE was compared directly with the medium-PE treatment, there was also no significant difference in hunger, fullness, satisfaction or thoughts of food as measured by VAS (P > 0.01).The mean VAS ratings for hunger, fullness, satisfaction and thoughts of food measured throughout each study day on the 4 treatment arms are shown in Figure ad libitum lunch are presented in Table Post-hoc analyses revealed that significantly greater energy was consumed at lunch when the two PE lipid treatments  were combined and compared with the no-PE control lipid , however there was no significant difference between the high-PE  and medium-PE  treatments. When composition of the lunch was examined across the 4 treatments there were significant differences between the intake of all 3 macronutrients  with mean intake at the lunch meal and conclude that overeating did not occur to any significant degree. Previous trials have described over-consumption when ~7 MJ was eaten at a single meal in participants with predicted energy requirements comparable to that of the subjects in our current trial [Despite its relationship with the known anorectic lipid OEA, there was no evidence from this intervention that the phospholipid PE altered perceptions of hunger and fullness or decreased food intake. This was a short-term assessment of appetite regulation in a group of lean men who arguably are likely to have good physiological regulation of appetite control. There are several issues that should be considered, including the appropriateness of the study design to detect relatively small changes in appetite control. There are a wide range of published methods by which appetite has been assessed and our current trial methodology was based on well known lipid emulsion trials -11. The Dietary fat is believed to be the least satiating of the 3 food macronutrients -3,20, anN-arachidonoylethanolamine (anandamide) and its analogue OEA [Fats and oils are neutral compounds. Replacement of a fatty acid within a triglyceride by a phosphate group results in a PL with 2 non-polar hydrophobic tails and a charged hydrophilic head. Two fatty acids are esterified to glycerol, which is linked through phosphate to a polar head group which in turn may comprise several different compounds; for example choline, serine or ethanolamine . A varieogue OEA .Rodriguez de Fonseca and colleagues demonstrated that food deprivation markedly decreased OEA biosynthesis in the small intestine, and thaThe dose of PE used in our study varied increased across treatments. The neutral lipid control contained little or no PL or PE, medium-PE treatment contained 1.96 g PL and 565.3 mg PE  and high-PE treatment contained 2.42 g PL and 682.4 mg PE . These doses were comparable with that of Rodriguez de Fonseca and colleagues who administered 5–20 mg/kg intraperitoneally  and NielIn conclusion, there was no evidence from this single dose trial that PE-containing phospholipids can modulate post-ingestive satiety and energy intake, even at relatively high doses. Whether PE may play a role when administered longer-term is not known.Eighteen healthy, non-smoking male adults were recruited via advertisement. Participants had no self-reported history of significant disease including cardiovascular disease, diabetes, or any other metabolic, endocrine or gastrointestinal disease. All were normotensive and had normal clinical biochemistry as assessed by lipid profile, full blood count and fasting blood glucose. None of the participants were taking medications which may have had an effect on appetite or weight regulation throughout the trial. Written consent was obtained from each participant and ethical approval obtained from the Northern Regional Ethics Auckland, New Zealand.This was a covert manipulation of dietary lipids which varied in their content of the phospholipid, PE. All participants in this randomised, cross-over were required to attend the Human Nutrition Unit on 4 occasions, each for a single day. Between treatment visits, participants returned home for a minimum of 5 days washout with the exception of 1 subject who completed his last visit with only a 1 day washout between treatments. During the washout period participants were requested not to make significant changes to their usual diet and exercise patterns wherever possible. Participants were also asked to abstain from alcohol and strenuous physical activity for 24 hours prior to the study-day, and to fast from 2000 h the previous evening. They completed a 24 h diet recall and 24 h physical activity questionnaire for the day prior to each treatment as a prompt to these requests. Participants were requested to arrive at the Unit using motorized transport and avoid morning exercise. Upon arrival, body weight and waist circumference were measured and any adverse events or medications were recorded. They were then given 200 ml water and completed the first series of visual analogue scales (VAS) which rated their subjective feelings of hunger, fullness, satisfaction and thoughts of food whilst faOn each study-day, participants received one of 4 test-breakfasts in randomised order: (i) high-PE phospholipid, (ii) medium-PE phospholipid, (iii) no-PE control neutral lipid, (iv) water control. Each of the 3 lipid treatments were matched for energy 2.5 MJ) and fat content (53 en%). The 0 kJ water control was used to confirm that the test system was sensitive to changes in energy. The test-breakfasts comprised 2 savoury muffins plus 300 ml water, or a 300 ml water control. The breakfasts were designed using the dietary program FoodWorks™  and the macronutrient composition, PL and PE content of the 4 breakfasts is shown in Table .5 MJ andThe high-PE treatment contained 2.42 g PL, of which 682.4 mg was PE which was equivalent to 9.75 mg/kg in a 70 kg individual. The medium-PE treatment contained 1.96 g PL, of which 565.3 mg was PE which was equivalent to 8.08 mg/kg in a 70 kg individual. Equivalent dose levels in animal trials have been shown to decrease spontaneous EI in rats ,18VAS were used according to the standard methodology of Flint and colleagues . The folad libitum lunch consisted of a restricted buffet-style meal with predominantly cold and one hot meal choice, along with a selection of beverages as shown in Table The ad libitum lunch were analysed using the dietary program FoodWorks™ . Single factor ANOVA  was used to determine any differences in EI or physical activity level on the day prior to each treatment visit. Single factor ANOVA  was also used to identify any differences in body weight at baseline between the 4 treatments or any changes in body weight, BMI or waist circumference throughout the trial. VAS data were analysed using repeated measures Linear Mixed Model ANOVA . The subject, the dietary treatment, the intervention period, and the study day were included in the procedure, as was the treatment/time interaction which addressed whether the trajectory over time during the intervention period differed between treatments (diet*time). Ad libitum lunch data was analysed using univariate ANOVA . Statistical significance was based on 95% limits (P < 0.05).Energy, macronutrient intake and the weight of the food consumed at the N-acylethanolamine; OEA: oleoylethanolamide; PE: phosphatidylethanolamine; PC: phosphatidylcholine; PL: phospholipid; PPAR-α: polyunsaturated fatty acids; PUFA: peroxisome-proliferator-activated receptor-α; SFA: saturated fatty acids; VAS: visual analogue scaleCHO: carbohydrate; EI: energy intake; HF: high fat; LCT: long chain triglyceride; MCT: medium chain triglyceride; MUFA: monounsaturated fatty acids; NAE: The authors declare that they have no competing interests.FEL protocol supervision, patient recruitment, registration, data collection, data entry, co-senior author. CMS data collection, data entry, manuscript preparation. ATM physician, clinical oversight of the participants, manuscript preparation. AKM development and preparation of the PE product. BHM biostatistician. SDP fundraiser, protocol design, study oversight, co-senior author.


The purpose of this study was to evaluate associations between long-term physical and psychological outcome variables in patients who survived meningococcal septic shock (MSS) in childhood.The study population was made up of all MSS patients requiring intensive care treatment between 1988 and 2001.th percentile of the reference groups on questionnaires to screen for psychopathology) (16/114) and 4) total intelligence quotient < 85 (18/115). No differences were found between patients with major physical sequelae and patients without major physical sequelae as to the presence of problem behaviour or total IQ < 85. Also, no differences were found between patients with mild neurological impairments and patients without as to the presence of problem behaviour or total IQ < 85. Finally, no differences were found between patients with major physical sequelae and patients without as to the presence of mild neurological sequelae. Less favourable scores on behavioural and emotional problems were significantly associated with poorer health-related quality of life (HR-QoL). HR-QoL scores were to a lesser amount predicted by severity of illness at time of PICU admission or by adverse physical outcome.A total of 120 patients visited the follow-up clinic (age at paediatric intensive care unit (PICU) admission 3.1 years; follow-up interval 9.8 years; age at follow-up 14.5 years ). Four major outcomes were considered: 1) major physical sequelae (defined as major scars and/or orthopaedic sequelae) (29/120), 2) mild neurological impairments (39/120), 3) problem behaviour (defined as a total score above the 90Long-term adverse physical and psychological outcomes in survivors of MSS did not seem to be associated. Poorer HR-QoL was mainly predicted by problem behaviour. The research presented here is part of a medical and psychological follow-up study of all consecutive surviving patients with meningococcal septic shock (MSS) requiring intensive care treatment between 1988 and 2001 at the Erasmus MC-Sophia Children's Hospital, Rotterdam, The Netherlands. This follow-up study revealed that these patients suffered from mild to severe long-term skin scarring, orthopaedic sequelae and neurological impairments. These patients had significantly higher severity of illness scores than patients without sequelae. Furthermore, they were assigned significantly poorer HR-QoL  scores, compared with normative data, though mainly on the physical domains. Overall, behavioural/emotional outcomes and cognitive functioning were normal .Until now associations between different outcome variables in MSS survivors have not been investigated. We hypothesize that adverse outcome variables are clustered together, not independently distributed. So patients suffering from severe skin scarring or extensive amputation would also show more problem behaviour and cognitive dysfunctioning. Furthermore we hypothesize that they show poorer HR-QoL.The present study is by no means a replicate of data already published. Indeed there are points of similarity  among previously published papers in our present study. However, in the present study our primary aim was to evaluate associations between long-term outcome variables, both physical and psychological, in patients who survived MSS in childhood. This knowledge would enable us to identify risk for overall adverse long-term outcome. The secondary aim was to assess various putative determinants of adverse overall outcome and HR-QoL.Patients were recruited from the PICU of the Erasmus MC-Sophia Children's Hospital, a tertiary care university hospital. Eligible for inclusion were all consecutive surviving patients aged 1 month to 18 years with a clinical picture of MSS, as well as their parents. Meningococcal septic shock was defined as septic shock with petechiae and/or purpura . The EraDuring the study period patients consecutively admitted with MSS were included in several sepsis studies -11. SeveParents and patients were interviewed by one paediatrician (CB) in a semi-structured format using a standard questionnaire with regard to health consequences since MSS. Complaints were defined as chronic if they developed after MSS and were still present at the time of the visit to the follow-up clinic. The same paediatrician physically examined the patients.Fifty-eight of the 120 patients (48%) had skin scarring due to purpura (ranging from barely visible scars to extremely mutilating scars); 10 (8%) amputation(s) of extremities (ranging from one toe to both legs and one arm); 7 (6%) lower limb-length discrepancy; 42 35%) neurological impairment(s) ; and 1(6%) of the 16 patients with septic shock-associated acute renal failure at PICU admission showed signs of mild chronic renal failure [5% neurolPatients were interviewed and examined by one psychologist (LV) using standard assessment procedures. Intellectual functioning was assessed with two tests for different age ranges: the Wechsler Intelligence Test III (WISC III) for the 6 to 15-year-olds; the Groninger Intelligence Test 2 (GIT2) for the 16 to 31-year-olds . OverallBehavioural and emotional problems were assessed with the Child Behaviour Checklist (CBCL) for the 6 to 18-year-olds, completed by the parents  and with the Adult Self-Report for the 18 to 31-year-olds patients was used  . Overallth percentile of the cumulative frequency distribution of the reference groups on a) the CBCL or b) the ASR. Youth Self-Report (YSR) ratings of patients aged 11 to 17 years also served to assess behavioural and emotional problems [Scores in the deviant range reflect levels of problem behaviors similar to those of children, adolescents and young adults typically referred for mental health services. Problem behaviour (a dichotomous variable) was defined as a total problem score above the 90problems .HR-QoL of patients < 18 years was assessed with the Child Health Questionnaire (CHQ); that of patients ≥ 18 years with the Short-Form 36 -24. SignStatistical analysis was performed with SPSS 12.0 for Windows .Comparisons between participating patients and non-participants were made with the Mann-Whitney test for age at time of PICU admission, length of stay in PICU and severity of illness scores; with the Chi-Square test for sex.We dichotomised and coded outcome variables (presence or absence of outcome variable), and then generated four major outcome variables; 1) major physical sequelae (n = 29/120) defined as major scars and/or amputation of extremities and/or limb-length discrepancy; 2) mild neurological impairments (n = 39/120) defined as hearing loss and/or chronic headache and/or focal neurological signs; 3) problem behaviour (n = 16/114); and 4) total IQ < 85 (n = 18/115). The latter category included three patients with mental retardation and epilepsy whose intelligence score was estimated to be < 70.Associations between these major outcomes  were evaluated with the Chi-Square test.The psychological outcome variables were also used as continuous variables. In that case the Mann-Whitney test was used to compare these psychological outcome scores between patients with and without major physical sequelae, as well as patients with and without neurological impairments.Adverse overall outcome was defined as adverse outcome on one or more of the four major outcome variables. The Mann-Whitney test was used to compare age at the time of PICU admission, length of stay in PICU and severity of illness scores between patients with and without adverse overall outcome.We tested the association between putative predictor variables  and long-term HR-QoL scores by using Spearman correlation for continuous variables and Mann-Whitney test for dichotomous variables. This was only done for those HR-QoL scales for which there were significant differences (poorer or better scores) between the study population and the normative data.P-value of 0.05 (two-sided) was considered the limit of significance.In all of the above mentioned statistical analyses, a Multiple linear regression analyses were applied to evaluate the predictive value of patient characteristics at the time of PICU admission on long-term HR-QoL scores. This was only done for HR-QoL scales if there were significant differences between the study population and the normative data.P-values of predictors were set to a level of 0.1 in the univariate analysis for entry in the regression analysis. Using backward elimination, independent predictors were identified with a P-value < 0.05. Continuous predictors with negative regression coefficients were considered as negatively associated with HR-QoL scales, those with positive values as positively associated.In the regression analysis, we included patient characteristics , disease variables  and follow-up interval. The target population consisted of 179 patients. Nine were lost to follow-up: one patient with severe adverse outcome  died several years after the MSS; seven had moved abroad; one was untraceable. Of the remaining 170 eligible patients, 145 agreed to participate. The other 25 patients and/or parents did not respond to the invitation or refused participation on practical or emotional grounds. Eventually, 120 patients visited the follow-up clinic. The median follow-up interval was 9.8 years (range 3.7 to 17.4 years), the median age of patients at the time of visit to follow-up clinic 14.5 years (range 5.3 to 31.1 years). Twenty-five patients and/or parents did not want to visit the follow-up clinic on practical  or emotional  grounds and preferred to fill in the questionnaires at home. The overall response rate, excluding patients lost to follow-up, was 71% (120/170). To check for possible selection bias, we compared characteristics of participants and non-participants (Table Neisseria meningitidis was cultured in blood. Seventy-eight of these (79%) had NM serogroup B, 13 (13%) serogroup C and in 8 (8%) the serogroup was not determined.At PICU admission a causative organism was isolated in 100 of the 120 patients (83%) who visited the follow-up clinic. In 99 patients (83%) Seventy-three of the 120 patients (61%) had adverse outcome on one or more of the four major outcome variables. Forty-seven of these 73 patients had adverse outcome on one major outcome variable: major physical sequelae n = 13, mild neurological impairments n = 19, problem behaviour n = 7 and total IQ < 85 n = 8.and mild neurological impairments n = 8, major physical sequelae and problem behaviour n = 2, major physical sequelae and total IQ < 85 n = 4, mild neurological impairments and problem behaviour n = 4, mild neurological impairments and total IQ < 85 n = 5, major physical sequelae, mild neurological impairments and problem behaviour n = 2, mild neurological impairments, problem behaviour and total IQ < 85 n = 1.Twenty-six of these 73 patients had adverse outcome on two or three major outcome variables: major physical sequelae The patient with chronic renal failure had amputation of a leg (below-knee), major scars and focal neurological signs. One of the three patients with mental retardation (estimated IQ < 70) had major scars and amputations; another had major scars and lower limb-discrepancy of 13 centimeters.There were no significant associations between the presence of the four major outcome variables. No differences were found between patients with major physical sequelae and patients without major physical sequelae as to the presence of problem behaviour or total IQ < 85. Also, no differences were found between patients with mild neurological impairments and patients without as to the presence of problem behaviour or total IQ < 85. Finally, no differences were found between patients with major physical sequelae and patients without as to the presence of mild neurological sequelae.P = 0.003, 4 versus 2 days) and higher severity of illness scores    compared with the 47 patients without adverse outcome.The 73 patients with adverse outcome had significantly longer length of stay in PICU  and long-term mild neurological impairments . As to tSo in our study group it seems that adverse long-term physical and psychological outcomes in survivors of MSS were not related. However, some specific cases should be mentioned, but they were insufficient to result in statistically significant differences: for example, two of the three patients with mental retardation (estimated IQ < 70) had major physical sequelae; the patient with chronic renal failure had major physical sequelae and focal neurological signs.et al. in children 4 to 10 years after bacterial meningitis [Long-term poorer HR-QoL was mainly predicted by less favourable scores on behavioural and emotional problem scales. HR-QoL scores, both on the physical and psychosocial domains, were significantly associated with problem behavior , regardless of age and parent-report versus patient-report. These findings are in line with those from a study by Koomen ningitis . This cophysical functioning. Indeed, amputation or limb-length discrepancy often resulted in important long-term morbidity  in our study group [general health perception was not significantly associated with the presence of major physical sequelae or mild neurological impairments. The significantly poorer scores on this HR-QoL scale, therefore, most likely reflected future health status, rather than the present one.HR-QoL scores were to a lesser amount significantly associated with adverse physical outcome; patients with major physical sequelae had significantly poorer scores on the HR-QoL scale dy group . SurprisStudies regarding long-term HR-QoL in survivors of other severe illnesses, for example, congenital heart diseases, also demonstrated weak associations between present physical health status and HR-QoL ,30.s< 0.7) on a minority of HR-QoL scales only in univariate analysis, the severity of MSS in childhood, regardless of any adverse outcome, seemed less important for long-term HR-QoL.Seeing that DIC and VAS, but not PRISM, showed small to moderate correlations  in one centre. The response rate was not high (71%), but we believe the results are valid since participating patients and non-participants did not differ with respect to age at time of PICU admission and severity of illness. It should be emphasized that only the most critically ill patients, that is, MSS patients requiring intensive treatment, were included. Therefore our findings cannot be extrapolated to the milder cases (sepsis) admitted to a general ward. Finally, baseline assessments of health status, psychological functioning and HR-QoL (before MSS) were not available, which would obviously be difficult to measure reliably under such stressful circumstances.Long-term adverse physical and psychological outcome variables were independently distributed, not clustered together. For example, patients suffering from severe skin scarring or extensive amputation did not show more problem behaviour and cognitive dysfunctioning compared with patients without severe skin scarring or extensive amputation. Poorer HR-QoL was mainly predicted by problem behaviour.More conclusive evidence could be obtained from a study comparing MSS survivors with survivors of other critical illness, matched on age and follow-up interval.• Seventy-three of the 120 patients (61%) had adverse outcome on one or more of the physical and psychological outcome variables.• The 73 patients with adverse outcomes had significantly longer length stays in PICU and higher severity of illness scores compared with the 47 patients without adverse outcomes.• Patients suffering from severe skin scarring or extensive amputation did not show more problem behaviour and cognitive dysfunction compared with patients without severe skin scarring or extensive amputation.• Patient's characteristics at the time of PICU admission revealed no significant associations with HR-QoL scores.• Concerning the physical and psychological outcome variables, HR-QoL scores were mainly related with problem behaviour.Neisseria meningitidis; PICU: paediatric intensive care unit; PRISM: Pediatric Risk of Mortality Score; VAS: vasopressor score; YSR: Youth Self-Report.ASR: Adult Self-Report; DIC, CBCL: Child Behaviour Checklist; CHQ: Child Health Questionnaire; Disseminated Intravascular Coagulation score; HR-QoL: health-related quality of life; MSS: meningococcal septic shock; NM: The authors declare that they have no competing interests.CMPB initiated this study and created the database, performed the statistical analysis and wrote the manuscript. LCACV assisted in creating the database, the interpretation of the results and writing of the manuscript. HR assisted in interpretation of the results and critically read the manuscript; JAH critically read the manuscript and assisted in interpretation of the results. WCJH performed the statistical analysis and assisted in the interpretation of the results and writing of the manuscript. EMWJ assisted in the interpretation of the results and writing of the manuscript. KFMJ initiated this study and assisted in the interpretation of the results and writing of the manuscript.