Construction workers in South Africa are regarded as a high-risk group in the context of HIV/AIDS. HIV testing is pivotal to controlling HIV transmission and providing palliative care and AIDS-related knowledge and stigma are key issues in addressing the likelihood of testing behaviour. In exploring these issues, various studies have employed an 11-item AIDS-related knowledge scale  and a 9-item stigma scale , but little evidence exists confirming the psychometric properties of these scales.Using survey data from 512 construction workers in the Western Cape, South Africa, this research examines the validity and reliability of the two scales through exploratory and confirmatory factor analysis and internal consistency tests.χ2 /df ratio = 1.675, CFI = 0.982, RMSEA = 0.038, and Hoelter (95 %) =393). A revised 8-item stigma scale was also developed  = 380). Both revised scales demonstrated good model fit and all factor loadings were significant (p < 0.01). Reliability analysis demonstrated excellent to good internal consistency, with alpha values of 0.80 and 0.74, respectively. Both revised scales also demonstrated satisfactory convergent and divergent validity. Limitations of the original survey from which the data was obtained include the failure to properly account for respondent selection of language for completion of the survey, use of ethnicity as a proxy for identifying the native language of participants, the limited geographical area from which the survey data was collected, and the limitations associated with the convenience sample. A limitation of the validation study was the lack of available data for a more robust examination of reliability beyond internal consistency, such as test-retest reliability.From confirmatory factor analysis, a revised 10-item knowledge scale was developed (The revised knowledge and stigma scales offered here hold considerable promise as measures of AIDS-related knowledge and stigma among South African construction workers. This finding aligns with Kalichman et al. (2005) in respect of the stigma scale, but no similar evaluation by them was done for the knowledge scale , but no association was found between HIV status and AIDS-related stigma scores.HIV+ workers scored significantly lower on the AIDS knowledge scale , but again no association was found between HIV testing and AIDS-related stigma scores.Workers who had previously tested for HIV/AIDS scored significantly higher on the AIDS knowledge scale [Tested  and Not Tested  with which they are assumed to be logically related  and each of them also successfully differentiated between groups of individuals who differed significantly in terms of their agreement on whether or not those with HIV should hide their status, agreement on whether or not they would rather not know if they were HIV positive, whether or not they felt they would be abandoned if they were to be infected with AIDS , whether or not they had been tested for HIV/AIDS, and their HIV serostatus. Where applicable, given the existence of previous research, these results correspond favourably with earlier findings .Overall, the results confirm the underlying reliability and validity of the two revised scales. However, this validation study has some limitations. Internal consistency was used as the sole measure of scale reliability. Practical limitations precluded the use of other forms of reliability - particularly test-retest reliability - that would have arguably provided a more robust test of the scales. The convergent and divergent validity tests, while strongly supportive of the scales, would undoubtedly have benefitted from the use of criteria such as behaviourial measures rather than only attitudinal or demographic variables.Additionally, the original study from which the data was obtained has some limitations. Firstly, the survey instrument failed to account for the choice exercised by respondents in terms of the specific language version of the questionnaire that they completed. As was clearly demonstrated, discrepancy between the test language and the native language of the ‘Black’ African respondents adversely affected the reliability of the revised AIDS knowledge scale for the entire sample. For the revised stigma scale, the results were less clear, suggesting a slightly higher reliability for ‘Black’ African respondents who completed the survey in their native language as compared to respondents from other ethnic groups. The overall direction suggests that the stigma scale might be far more reliable in communities that favour traditional views and beliefs about AIDS and HIV as compared to communities where less traditional and more scientific explanations are favoured. However, as the magnitude of the difference observed for the stigma scale was marginal, it is not advisable to read too much into this finding.The native language-test language variation issue should nevertheless be flagged for special attention in further research, particularly that conducted in multilingual settings. In this regard four issues are critical: 1) the actual choice exercised by respondents in terms of the test language for the completion of the questionnaire, 2) attention to translation methods for multilingual versions of the scales, to ensure greater conceptual correspondence between translated versions, and 3) a more direct examination of how the overall sample reliability estimates may be masking significant differentiation in such estimates in language-defined sub-samples. Additionally, it is recommended that the language analysis also be extended to validity testing, as it is likely that the findings of variable scale reliability estimates are derivative of variable scale validity as a function of language.Furthermore, the data from the original study were obtained from construction workers on sites in the greater Cape Town metropolitan area, and are not therefore representative of the population of on-site construction workers in other provinces, or the country as a whole. Given the variability in ethnicity, language and cultural beliefs and values across different regions of the country, it remains for future research to determine whether or not the dimensionality and reliability of the scales as established here will prove invariant in different samples, not only for construction workers but also for the communities from which these workers emanate.Finally, the limitations associated with convenience sampling need to be acknowledged. Participating workers employed by co-operating construction firms who have previously participated in HIV/AIDS research are likely to be more knowledgeable about HIV/AIDS, and are less likely to be prejudicial towards and discriminatory against HIV+ persons.In the context of HIV/AIDS, construction workers in South Africa are regarded as a high-risk group. HIV testing is pivotal to controlling HIV transmission and providing palliative care and AIDS-related knowledge and stigma are key issues in addressing the likelihood of testing behaviour. This study examined the psychometric properties of the 11-item knowledge and 9-item stigma scales developed by Kalichman and Simbayi (2004)  and Kali


Clostridium difficile infection (CDI), but little is known about how risks cumulate over the course of therapy and abate after cessation. We prospectively identified CDI cases among adults hospitalized at a tertiary hospital between June 2010 and May 2012. Poisson regression models included covariates for time since admission, age, hospitalization history, disease pressure, and intensive care unit stay. Impacts of antibiotic use through time were modeled using 4 measures: current antibiotic receipt, time since most recent receipt, time since first receipt during a hospitalization, and duration of receipt. Over the 24-month study period, we identified 127 patients with new onset nosocomial CDI . Of the 4 measures, time since most recent receipt was the strongest independent predictor of CDI incidence. Relative to patients with no prior receipt of antibiotics in the last 30 days (IR = 2.95), the incidence rate of CDI was 2.41 times higher  during antibiotic receipt and 2.16 times higher when patients had receipt in the prior 1–5 days . The incidence rates of CDI following 1–3, 4–6 and 7–11 days of antibiotic exposure were 1.60 , 2.27  and 2.10  times higher compared to no prior receipt. These findings are consistent with studies showing higher risk associated with longer antibiotic use in hospitalized patients, but suggest that the duration of increased risk is shorter than previously thought.Antibiotic therapy is the principal risk factor for  Clostridium difficile infection (CDI) is a hospital and community-acquired disease that especially impacts elderly hospitalized patients receiving antibiotics C. difficile spore germination Antibiotic receipt represents the most important known risk factor for CDI; it is thought to induce CDI risk by denuding the gut of protective bacteria and increasing Study approval was obtained from the Research Ethics Board of Sunnybrook Health Sciences Centre who waived the need for patient consent because there was no contact with the patients and anonymity was assured.A cohort study design was used to assess the association of antibiotic exposure with the incidence of CDI among patients admitted to Sunnybrook hospital, a large acute care teaching hospital located in Toronto, Canada. The source cohort consisted of all patients over 18 years old, without a previous CDI diagnosis, and hospitalized in an acute care ward at Sunnybrook hospital in the June 1, 2010 to May 31, 2012 period and excluded time spent in the hospital's psychiatry ward.C. difficile infected patients were prospectively identified by the Infection Prevention and Control (IPC) department via active surveillance during the study period. In accordance with the provincial surveillance definition For CDI cases, event time was the number of days from hospital admission to symptom onset, or positive toxin assay for rare cases in which symptom onset was missing. For non-cases, censoring time was the number of days from hospital entry until discharge, study termination, or death.th edition Patient antibiotic exposures were drawn from pharmacy dispensing records. We examined for daily receipt of any antibiotic but excluded exposure to metronidazole and oral vancomycin since these may be treatments for CDI. Daily antibiotic receipt was classified according to the Anatomical Therapeutic Chemical (ATC) Classification System, 17Using the antibiotic classes identified above, we developed two alternative measures of antibiotic exposure: (1) an index representing the number of distinct classes of antibiotics a patient received on a given day , and (2) a categorical antimicrobial risk index based on Brown et al. We created 4 variables based on patients' unique antibiotic exposure histories: (1) current antibiotic receipt, which was dummy coded as 1 for days when a patient received an antibiotic, and 0 otherwise, (2) the time lapse since the most recent antibiotic use (in days), which was categorized as 0 (current use), 1–5, 6–30, >30 (including no use), (3) time lapse since the start of the first antibiotic exposure during a given hospitalization, which was categorized as 0–2, 3–6, 7–14, 15–30 and >30 (including no use), and (4) the duration of antibiotic use, categorized as 0 (no use), 1–3, 4–6, 7–11 and >11.after symptom onset to 14 days after the positive test associated with case detection. This is in keeping with Dubberke et al. Patient age, sex, hospital pharmacy record, and bed assignment, were obtained from electronic hospital administrative records. Infection pressure was derived by calculating the number of diagnosed infectious patients with CDI in each ward of the hospital at noon of each day using patient location records. The infectious period of a given diagnosed patient  was defined as starting on the day For bivariate analyses in which we compared characteristics of the at-risk period of cases with a 10% simple random sample of non-case patients (controls), two-sided p-values were assessed with Pearson's chi-squared test for categorical variables and with the Wilcoxon rank-sum test for continuous variables. For the principal case-cohort analysis based on Poisson regression (models described below), which were used to measure unadjusted incidence rates and unadjusted and adjusted incidence ratios, we weighted all of the control patients' follow-up times by 10 and all of the case patients' follow up times by 1, so as to reflect rates from the original, complete, cohort, as per the Barlow method To estimate the impact of antibiotic exposures on CDI risk, we developed weighted Poisson regression models that aimed to predict the time elapsed from hospital admission to the occurrence of a first CDI infection. Our data was structured in counting process format with one record for each patient-day. For each of the 4 antibiotic exposure covariates, two models were fitted to the data. The 4 unadjusted models included no covariates other than antibiotic exposures; incidence rate ratios were estimated using Poisson regression. The 4 adjusted models included 6 potential confounders: time since admission (modeled as a b-spline with knots at 5 and 15 days), patient age , sex, number of previous hospital admissions , patient-days of infection pressure in the past 10 days, and ICU admission in the past 10 days. The number of adjustment factors was restricted in order to ensure at least 10 events per covariate Subsequently, we used the best fitting of the 4 antibiotic exposure covariates to determine risk associated with the two risk indexes and for each of the 8 antibiotic classes. For unadjusted and adjusted estimates of the antibiotic-specific risks, antibiotic exposure adjustment variables were derived which measured exposure to any other antibiotic without exposure to the antibiotic in question.Analyses were conducted using R statistical software (v3.0.2); the glm and geeglm C. difficile PCR test date rather than symptom onset date to define the outcome timing. Also, because pre-admission antibiotic exposure information was not available, we conducted an additional sensitivity analysis excluding cases and patient time in the first 10 days of each hospitalization to investigate the impact of exposure history incompleteness.In order to assess the potential impact of uncertainty of diagnostic timing on the analyses, we conducted a sensitivity analysis using positive Over the two-year study period, and before exclusion of ineligible patients, a total of 47,241 patients were identified as having been admitted to Sunnybrook Health Science Centre . Of thesThe median age of cases (72.0) was almost 5 years older than that of controls . The majority (71.7%) of cases received a cephalosporin class of antibiotic compared to 33.1% of controls (p<0.001). Similarly, penicillins and fluoroquinolones were more likely to be prescribed among case patients compared to controls. Among case patients, median symptom onset date was 9 days after admission (IQR: 5–17 days).The incidence of CDI when patients received antibiotics was 8.43 per 10,000 days  compared to 4.47  when patients did not receive antibiotics ; it follAmong the 4 antibiotic exposure measures, the time since last antibiotic use was the most important independent predictor of CDI onset, and yielded a statistically significant improvement in the prediction of CDI onset (Δ adjusted AIC = −4.6). The incidence of CDI  was 2.95 when patients had received no antibiotics in the previous 30 days (reference), 8.43 when patients were currently receiving antibiotics , 8.12 when patients had recent receipt in the last 1–5 days , and 3.58 when patients had received antibiotics in the last 6–30 days . The observed association was highest 3–6 days  and 7–14 days  after the start of antibiotics. The duration of antibiotic exposure yielded a worse model fit relative to the adjusted model with current antibiotic use (Δ adjusted AIC = 5.1).For all remaining analyses including the sensitivity analyses and the investigation of antibiotic class-specific effects, we categorized antibiotic exposure history as receipt of antibiotics in the last 5 days (which included current receipt), or no receipt in the last 5 days. The adjusted risk was 2.35  for patients that had received an antibiotic in the previous 5 days . We condIn order to consider differences in the level of risk among antibiotic users, we considered risk among patients with exposure to various combinations of antibiotics and to specific antibiotics . The adjrd & 4th generation cephalosporins taken alone or in combination, had higher risk in comparison to other antibiotic classes .For each of the 8 antibiotic classes and subclasses for which there was sufficient exposure for individual analysis (>2% of subcohort patient-time), we measured the risk of the given antibiotic taken alone or in combination with other antibiotics, relative to no antibiotic exposure. In adjusted analyses, all 3 of the most prescribed antimicrobial classes demonstrated similarly large hazard ratios. Of the subclasses, 3Our observational study of 127 CDI cases and 2 years of follow-up on patients at a large tertiary hospital found that: (1) in-hospital CDI risk was highest 3 to 14 days after the start of the first antibiotic course, (2) elevated CDI risk persisted for a period of 5 days after the end of antimicrobial therapy, and (3) patients with longer antibiotic courses were at higher risk of developing CDI than patients with shorter courses, but even short courses and single doses of antibiotics incur a substantial risk of inducing CDI.Our study is the first to compare the duration of antibiotic use, time since antibiotic initiation, and time since antibiotic cessation as measures of CDI risk; our results showed that measuring the time since last antibiotic use is the most predictive metric for quantifying risk for a patient, whereby risk during and within 5 days of cessation was the most elevated. Antibiotic duration has already been shown to be more predictive than cumulative dosage for predicting CDI risk A strength of this study was our use of a combination of analytic methods tailored to the study of nosocomial infections. Foundational texts in epidemiologic methods principally covered the use of logistic regression and survival analysis for the study of chronic diseases that develop over the course of many years C. difficile diagnoses. Considering discharged patients as censored surmounted this limitation, but means that our results are most generalizable to acute, hospital-onset CDI. Furthermore, clinical teams are aware of antimicrobial exposures which may prompt diagnostic testing for CDI C. difficile colonization status and timing of acquisition of the organism, which would be expected to influence the time lapse between antibiotic exposure and disease onset. Finally, our hospital information system lacked information on patient comorbidities and other healthcare exposures including discharges and outpatient visits to other hospitals in the region.A limitation of our study was our lack of information on outpatient antimicrobial exposures prior to patient hospitalization, since our exposure free reference group used for calculating hazard ratios could have included patients exposed to antibiotics prior to admission. However, our findings were robust in sensitivity analyses considering subsets of patients with more prolonged hospitalization and therefore more complete antibiotic exposure histories. Further, our study lacked information on post-discharge Antibiotic use is the most important risk factor for CDI, and a substantial amount of research has considered the risk of CDI associated with different antibiotic exposures. In this study of the association between antibiotic exposures and CDI risk in hospitalized adults, we focused on the timing of increased risk; we found that risk appears 3 days after the onset of antibiotic use, and continues for a period of 5 days after the end of antibiotics, and is relatively unimportant thereafter. Further research may consider how different antibiotics may induce different time-varying risks in order to differentiate antibiotic impacts and improve patient outcomes.Appendix S1R Statistical Software Analysis Code For Tables and Poisson Regression Models.(TXT)Click here for additional data file.


Nonomuraea sp. JAJ18 were carried out using combination of both nonstatistical one-factor-at-a-time (OFAT) method and statistical response surface methodology (RSM). Using OFAT method, starch and (NH4)2SO4 were identified as suitable carbon and nitrogen sources, respectively. Subsequently, starch, NaCl, and MgSO4·7H2O were recognized as the most significant media components with confidence level of above 95% using the Plackett-Burman design. The levels of the three media components were further optimized using RSM employed with Box-Behnken design. Accordingly, a second-order polynomial regression model was fitted into the experimental data. By analyzing the response surface plots as well as using numerical optimization method, the optimal levels for starch, NaCl, and MgSO4·7H2O were determined as 15.6 g/L, 0.8 g/L, and 1.98 g/L, respectively. With the optimized medium, 15.5% increase was observed in antibiotic activity of JAJ18. Results further support the use of RSM for media optimization. To the best of our knowledge, this is the first report of statistical media optimization for antibiotic production in rare actinomycete Nonomuraea species, which will be useful for the development of Nonomuraea cultivation process for efficient antibiotic production on a large scale.The selection and optimization of nutritional constituents as well as their levels for the improved production of antibiotic by  Since the report of streptomycin, actinomycetes, especially streptomycetes, have been shown to be a prime source of antibiotics. However, the likelihood of finding novel compounds has dwindled due to extensive studies on the ubiquitous species . In the Improving the antibiotic production is prerequisite to achieve adequate antibiotic yield for evaluating its potential and novelty. Antibiotic producing ability of the actinomycetes is not a static property and it is highly influenced by culture conditions and media components –9. There Nonomuraea sp. JAJ18, which was previously isolated from an Indian coastal solar saltern [The aim of the present work was to design and optimize cultural parameters as well as significant media components to achieve improved antibiotic production in rare actinomycete, saltern . The stu Nonomuraea sp. JAJ18 and the pure culture was maintained at 4°C on modified inorganic salt agar slants which contained 10 g of starch, 4.0 g of yeast extract, 20 g of NaCl, 2.0 g of (NH4)2SO4, 1.0 g MgSO4·7H2O, 1.0 g K2HPO4, and 22 g of agar in 1.0 L of distilled water. The bacterial indicator strain, Bacillus subtilis MTCC 441 used for antibiotic assay was maintained over Mueller Hinton agar slants. The MTCC culture was obtained from Microbial Type Culture Collection, IMTECH, India.The rare actinomycete strain JAJ18 used in this study was previously isolated from a coastal solar saltern established at Tuticorin, India . It was Fermentation broth of JAJ18 was centrifuged at 10000 rpm for 10 min and the cell-free supernatant was recovered. Ethyl acetate was added to the supernatant in 1 : 1 proportion and the mixture was agitated for 20 min. The solvent layer containing antibiotic substance was separated from broth and it was further centrifuged at 5000 rpm for 15 min to remove traces of fermentation broth. The crude extract was concentrated tenfold using a rotational vacuum concentrator and used for antibiotic assay. Bacillus subtilis MTCC 441 by agar diffusion plate assay [ B. subtilis suspension adjusted to a McFarland standard of 0.5, which is equivalent to 1.5 × 108 CFU/mL. A sterile disc impregnated with ethyl acetate was used as control. The plates were incubated at 37°C for 24 h and the inhibition zone formed around the disc was measured in millimetre. Maxwell et al. [Antibiotic assays were carried out in triplicates againstte assay , 17. Stel et al.  confirmeThe mycelial pellet was collected by centrifugation at 10000 rpm for 10 min and dried at 60°C. The dried pellet was weighed to determine the growth. The growth in terms of mycelial biomass accumulation was expressed as g/L of the culture medium.In preliminary experiments, various culture conditions, carbon sources, and nitrogen sources were evaluated for their suitability to sustain good antibiotic activity by strain JAJ18. An appropriate basal medium with chosen carbon and nitrogen sources was taken for statistical screening and further optimization using PBD and RSM, respectively.4)2SO4, 1.0 g of MgSO4·7H2O, and 1.0 of g K2HPO4 in 1.0 L distilled water at a rate of 108 spores in 50 mL medium. Cultures were kept on a shaker at 120 rpm at 30°C for 3 d and used as seed stocks.Spore suspension of JAJ18 was prepared in Milli-Q water from the culture grown on ISP4 medium supplemented with 4% of yeast extract and 0.5% of NaCl (w/v) at 30°C for 10 d. The suspension was added to seed medium which contained 5.0 g of starch, 5.0 g of glucose, 4.0 g of yeast extract, 5.0 g of NaCl, 2.0 g of  containing 400 mL of the basal medium was inoculated with seed culture and incubated at 30°C in shaking incubator at 120 rpm for 16 d. At 24 h interval, 25 mL of culture broth was collected and centrifuged at 10000 rpm for 10 min. The crude compound was extracted from the mycelia-free supernatant and tested for antibiotic activity against B. subtilis to determine the optimum pH for antibiotic production.A pH suitable for JAJ18 was determined using series of 250 mL Erlenmeyer flasks containing 50 mL of the basal medium adjusted to pH 5.5, 6, 6.5, 7, 7.5, 8, 8.5, and 9. Each flask was inoculated with 2% V/V of seed culture. All of them were incubated at 30°C for 10 d in shaking incubator at 120 rpm. Crude compound was extracted and antibiotic activity was tested against Nonomuraea sp. JAJ18 and incubated at different temperatures such as 20, 25, 30, 37, 40, 45, and 50°C for 10 d in shaking incubator at 120 rpm. Crude compound was extracted and antibiotic activity was assayed against B. subtilis to determine the optimum temperature for growth and antibiotic production from JAJ18.Similarly, for determination of optimum temperature, the Erlenmeyer flasks containing 50 mL of basal medium were inoculated withIn order to select the ideal carbon and nitrogen sources for the production of antibiotic from JAJ18, one-factor-at-a-time (OFAT) method was used. Various carbon  and nitrogen  sources were used individually instead of the corresponding carbon and nitrogen sources in the chosen basal medium while other components were kept constant at original concentration, and the antibiotic activity was determined after 10 d of incubation at 30°C in a rotary shaker with 120 rpm. Combination of each nitrogen source with 0.4% (w/v) yeast extract (YE) was also attempted. Nonomuraea sp. JAJ18. Minitab 15.0  was used for the development of experimental design and subsequent analysis of the experimental response data. Using the PBD, 8 medium components (independent variables) were screened by representing them at two levels o levels . The exp4·7H2O, for improving the antibiotic production by JAJ18, RSM was used with Box-Behnken design. The Design expert trial package (version 7) was used for the experimental design and the regression analysis of the data. The three medium components (independent variables) were taken as key input variables at three different levels, (−), (0), and (+) for low, intermediate, and high concentrations, respectively . The quality of the polynomial model equation was tested statistically by coefficient of determination (R2) and adjusted R2. The fitted polynomial equation was then expressed in the form of three-dimensional surface plots, to illustrate the relationship between the responses and the experimental levels of each independent variable. The Design Expert's numerical optimization method was employed to optimize the level of each variable for attaining maximum response.The statistical significance of the model was verified using the analysis of variance (ANOVA). Overall model significance was determined using Fisher's The combination of different optimized variables, which gave the maximum response, was experimentally validated by culturing JAJ18 in optimized and unoptimized production medium in shake flasks. The cell-free culture broths were collected by centrifugation and extracted with equal volume of ethyl acetate; the top organic layer was concentrated and assayed for antibiotic activity. Nonomuraea sp. JAJ18 in batch cultures.Selection of basal medium is a prefatory and significant step for further formulation of an optimized medium to enhance antibiotic production by actinomycetes. In order to select a basal medium, strain JAJ18 was subjected to antibiotic production in three different media and tested for antibiotic activity. Modified ISP4 medium showed a maximum antibiotic activity (120 U/mL), followed by ISP4 medium (90 U/mL), and yeast extract malt extract glucose medium (80 U/mL). This is in agreement with previously reported employability of modified ISP4 for antibiotic production from actinomycetes of saltern origin . ConsequCarbon and nitrogen sources are the vital constituents of culture media for better antibiotic production from actinomycetes , 21. EffLikewise, among the various nitrogen sources, higher antibiotic production was favoured with ammonium sulfate (116.6 U/mL) followed by yeast extract (103.3 U/mL). Lower antibiotic activities were observed with beef extract (83.3 U/mL) and peptone (96.6 U/mL). However, maximum antibiotic activity (133.3 U/mL) was observed with ammonium sulfate when supplemented with yeast extract. Thus, starch and combination of ammonium sulfate and yeast extract were chosen as the source of carbon and nitrogen for further experiments, respectively.4)2SO4, and yeast extract were used in production media as the major nutrients for the production of cyclic peptide antibiotics in Nonomuraea species [ Nonomuraea. However, starch has been reported to be the major carbon source for antibiotic production by other actinomycetes, for instance, gentamicin production by Micromonospora echinospora [ Streptomyces avermitilis using ammonium sulfate as the sole nitrogen source. Lee et al. [ Streptomyces hygroscopicus. Amending the production media with yeast extract has been widely reported to have significant influence on secondary metabolite production. In the present study, yeast extract was found to favour antibiotic production in JAJ18 which is in good agreement with a report which asserts that yeast extract was able to support relatively high antibiotic production in actinomycete, Streptomyces sp. IMV-70 [Starch, , NaCl (X4), and MgSO4·7H2O (X5) had significant effect on antibiotic production which was apparent from their confidence levels above 95% in comparison to other variables. It was further confirmed from Pareto chart  that affected the production of antibiotic by JAJ18. A batch of experiments was conducted according to Box-Behnken design and the results were summarised along with the predicted values (Y was the predicted response (antibiotic activity) and A, B, and C were the coded values of starch, KBr and CaCO3, respectively.Several researchers working on antibiotics discovery programs have applied PBD and RSM as statistical tools to recognize, manipulate, and optimize influencing medium constituents and recorded the increased antibiotic production , 34. RSMd values  to deterF-test with low P value (<0.0001). Significance of model was further supported by statistically insignificant lack of fit, as was evident from the lower calculated F-value (1.16). Accuracy of the model can be checked by the determination of coefficient of R2. The closer the value of R2 to 1, the stronger the model to predict the response [R2 of 0.9972 implied that model equation could explain 99.72% of the total variation in the response. The observed R2 value was comparable with the earlier reports [The analysis of variance (ANOVA) was performed to statistically inspect the quadratic regression model and the results are summarised in response . The mod reports , 35.In order to judge the model adequacy and clarify the signs of any problems in the experimental data, a series of diagnostic plots can be used . Plot of4·7H2O. The 3D response surface plots were drawn to illustrate the pairwise combination of the three variables, while the rest was held at middle level , the antibiotic activity gradually increased to a maximum at low concentration of NaCl , the antibiotic activity increased with increasing concentration of starch from 2 to 20 g/L . Further increase of MgSO4·7H2O to its high level  resulted in significant increase in antibiotic production even at lower concentrations of starch ; however, intensity of decrease was significantly reduced with increasing concentration of MgSO4·7H2O  plots assisted in understanding the main as well as the interaction effects of three factors, starch, NaCl and MgSOle level . It is o of NaCl . Howeverf starch . With thSO4·7H2O .4·7H2O = 0.99, which were 15.6 g/L starch, 0.8 g/L NaCl and 1.98 g/L MgSO4·7H2O, respectively.Using numerical optimization method, predicted maximum antibiotic activity was 154.49 U/mL , when th Nonomuraea sp. JAJ18. The final optimized medium contained 15.6 g of starch, 2.0 g of (NH4)2SO4, 4.0 g of yeast extract, 0.8 g of NaCl, 1.98 g of MgSO4·7H2O, 1 g of K2HPO4, 2 g of CaCO3, and 0.004 g of FeSO4·7H2O in 1 L of distilled water.In order to validate the statistical results, a verification experiment was performed in triplicate with a predicted optimal medium in shake flasks . The ant Nonomuraea sp. by medium optimization using a combination of nonstatistical and statistical methods. The study focused primarily on improved production of antibiotic by Nonomuraea sp. JAJ18 as a function of various carbon and nitrogen sources and levels of ingredients in production medium as well as culture conditions. The results support the use of nonstatistical one-factor-at-a-time method for recruitment of suitable carbon and nitrogen sources. Further, PBD and RSM were found to be very effective in determining and optimizing the medium components in manageable number of experimental trials with overall 15.5% increase in antibiotic activity. The optimum culture medium and the culture conditions defined from this study will be useful for further development of large scale fermentation process for the efficient production of antibiotic from this Nonomuraea sp. JAJ18.To the best of our knowledge, this is the first report on improved antibiotic activity of


Neoadjuvant chemo-radiotherapy (CRT) followed by surgical resection is the standard treatment for locally advanced rectal cancer, although complete tumor pathological regression is achieved in only up to 30% of cases. A clinicopathological and molecular predictive stratification of patients with advanced rectal cancer is still lacking. Here, c-Met and YKL-40 have been studied as putative predictors of CRT response in rectal cancer, due to their reported involvement in chemoradioresistance in various solid tumors.2) neoadjuvant CRT or the XELOXART protocol. CRT response was documented on surgical resection specimens and recorded as tumor regression grade (TRG) according to the Mandard criteria.A multicentric study was designed to assess the role of c-Met and YKL-40 expression in predicting chemoradioresistance and to correlate clinical and pathological features with CRT response. Immunohistochemistry and fluorescent in situ hybridization for c-Met were performed on 81 rectal cancer biopsies from patients with locally advanced rectal adenocarcinoma. All patients underwent standard (50.4 gy in 28 fractions + concurrent capecitabine 825 mg/mA significant correlation between c-Met and YKL-40 expression was observed (R = 0.43). The expressions of c-Met and YKL-40 were both significantly associated with a lack of complete response . Thirty of the 32 biopsies co-expressing both markers had partial or absent tumor response (TRG 2-5), strengthening their positive predictive value (94%). The exclusive predictive role of YKL-40 and c-Met was confirmed using a multivariate analysis . TRG was the sole morphological parameter associated with poor outcome.c-Met and YKL-40 expression is a reliable predictor of partial/absent response to neoadjuvant CRT in rectal cancer. Targeted therapy protocols could take advantage of prior evaluations of c-MET and YKL-40 expression levels to increase therapeutic efficacy. This in vitro ,37,40. Soblastoma. HoweverHere, we show that a) c-Met and YKL-40 are independent predictors of poor response to CRT and b) tumor regression grade (TRG), but neither c-Met nor YKL-40, is the sole prognostic marker of shorter overall and disease survival.2), whereas the remaining 30 patients (37%) underwent a XELOXART trial [Eighty-one rectal cancer pre-treatment endoscopic biopsies and their matched surgical specimens were retrieved from consecutive cohorts from the archives of the pathology divisions of three Italian Institutions between January 2006 and December 2012: University of Torino at Città della Salute e della Scienza (Molinette) Hospital of Turin 46 cases); San Luigi Hospital of Orbassano (16 cases); and Institute for Cancer Research and Treatment of Candiolo (19 cases). All patients had a locally advanced rectal adenocarcinoma eligible for neoadjuvant CRT (cT3-T4 stage). Fifty-one patients (63%) received CRT according to a standard capecitabine regimen -stained slides available from biopsies and surgical specimens were reviewed, and a representative paraffin block was selected for each case. Biopsy and surgical specimens of each patient were independently reviewed by two dedicated pathologists (RS and PC), who confirmed all diagnoses. On the rectal pre-treatment biopsies, the tumor grade (G1-G2-G3) and apoptosis were evaluated. Apoptosis was assessed by evaluating the apoptotic bodies lining the basal membrane, and it was scored as negative (<25% per 100 viable cells) or positive (> = 25% per 100 viable cells). Vascular invasion was not included among the histopathological parameters analyzed since biopsy samples are usually superficial and small-sized and therefore considered poorly reliable for the presence of neoplastic emboli. Response to neoadjuvant CRT was established by a histopathological examination of surgically resected rectal specimens using the tumor regression grade (TRG), according to Mandard criteria , which aImmunohistochemistry was performed in all but five cases of pre-treatment biopsies, which had insufficient tissue from the rectal biopsy. Resected surgical specimens with residual cancer (TRG2-5) were analyzed as well. Three ìm thick serial paraffin sections of each case were processed by immunohistochemistry using an automated immunostainer  with antibodies against YKL-40  and c-Met . The antigen retriewal step was included in the automated programme. A biotin-free, dextran chain-based detection system  and diaminobenzidine  were used as the chromogen, according to standard protocols. Neoplastic tissues were assembled on tissue microarrays, excluding the primary antibody and IgG-matched serum, and were used as positive and negative controls.All immunostained slides were analyzed independently by RS, AC and PC, who were blinded to the clinical data. YKL-40 and c-Met staining was assessed as both categorical (negative or positive if present in at least 1% of the neoplastic cells) and continuous variables (scored as number of positive neoplastic cells). When positive, cells had a moderate to strong cytoplasmic reactivity for both antibodies. In case of discrepancies, slides were reviewed using a multihead microscope, and a consensus was reached.al. [c-Met FISH analysis was performed using the probe mixtures set, MET/CEP7 , in all biopsies with a positive c-Met immunostaining. FISH experiments were carried out according to the manufacturer’s instructions as follows: 10 μl of probe mixture was applied to each slide, which was immediately covered with a coverslip and sealed with rubber cement. Slides were thoroughly co-denatured in the Hybrite System  at 73°C for 3 minutes and hybridized overnight at 37°C. A post-hybridization wash was performed in 2xSSC-0.3% NP-40 at 73°C for 3 minutes. Then, slides were dehydrated and counterstained with DAPI I . An automated scanning station, MethaSystems , equipped with an AxioImager–Z1 epifluorescence microscope was used to determine the c-Met gene status. Tumor areas with high signal quality and good nuclei preservation were assessed. These areas were automatically scanned, and 10 different consecutive focal planes were made for each FISH signal to form a single bidimensional image. For traditional reading, the automatically acquired images were transferred to the Isis software  and stored in dedicated files. FISH data were analyzed using previously described criteria, according to Capuzzo et al. .Correlations between YKL-40 and c-Met antibodies were made using the Chi-square test and two-tailed Spearman’s test when considered categorical or continuous variables, respectively. Clinico-pathological parameters for univariate analyses to determine predictors of the response to CRT included tumor grade, apoptosis, YKL-40 and c-Met staining patterns. Fisher’s exact or Chi-square tests were used. All parameters in the univariate analysis with a significant impact on predicting CRT response were considered for multivariate analysis using the Cox proportional hazard model. To analyze the prognostic impact of all pathological variables considered, the univariate overall survival analysis was based on the Kaplan–Meier product limit estimate of survival distribution. Unadjusted differences between survival curves were tested using the log-rank test. All tests were performed using the Statistical Package for the Social Sciences version 17 . A significance level of p<0.05 was used.Table 1.The main clinical and pathological features of the whole series of 81 cases are summarized in Fig 1).According to TNM staging, 20 cases (25%) had a post-CRT pathological T stage ypT0, 4 cases (5%) had ypT1, 20 cases had (25%) ypT2, 35 cases had (43%) ypT3 and 2 cases had (2%) ypT4. Lymph node metastases were detected in 22 cases (27%). The clinical T stage (cT) was obtained in 74 cases (91%), and a T-downstaging after RCT was observed in 48 (65%) of them. Applying the Mandard criteria to define the TRG, 20 cases (25%) had a complete pathological tumor response to neoadjuvant CRT (TRG1-ypT0) on the surgically resected rectal specimens. In the remaining 61 patients (75%), a partial or absent tumor response was observed, with 16 cases (20%) classified as TRG2, 22 cases (27%) classified as TRG3, 21 cases (26%) classified as TRG4 and 2 cases (2%) classified as TRG5  was similar in the two subgroups of treatment (RT+capecitabine or XELOXART protocol).The distribution of complete (TRG1) Fig 2A and 2B), while forty-three (56%) cases were positive for c-Met with a moderate or strong cytoplasmic reactivity .Overall, YKL-40 tumor expression was observed in 47 (62%) cases, with a moderate or strong immunostaining localized within the cytoplasm of neoplastic cells . The expression of YKL-40 and c-Met was significantly correlated when considered both continuous variables (r = 0.43 and p = 0.001) and categorical variables . YKL-40 was positive in moderately or poorly differentiated cases (p = 0.018) and in those lacking inflammatory infiltrate (p = 0.01), whereas c-Met was positively correlated with the absence of apoptosis (Table 2).Both YKL-40 and c-Met tumor expression was retained in resected rectal specimens according to their expression on endoscopic biopsies. c-Met FISH analysis was performed in all 43 c-Met-positive cases. No c-Met gene amplification was found, whereas nine (21%) cases demonstrated low/high gene polisomy .In a univariate analysis subdividing the entire series into complete (TRG1) or non-complete responders (TRG2-5), neither tumor grade nor the presence of apoptosis were detected on rectal pre-treatment biopsies with TRG, indicating that none of these histological features proved effective in predicting CRT response. Conversely, YKL-40 and c-Met were differentially expressed into the five TRG subclasses  and categorical variables  , and it had high positive predictive values  (Table 4).In the TRG1 subgroup, the YKL-40 tumor expression ranged from 0 to 30% , and that of c-Met ranged from 0 to 50% ; specifically YKL-40 and c-Met immunoreaction was absent in 67% and 62% of TRG1 cases, respectively. In the group of partial responders (TRG2-5), the YKL-40 expression varied from 0 to 80% , and that of c-Met ranged from 0 to 60% . Even when considered dichotomized variables, the expression of both biomarkers was significantly associated with a lack of complete tumor regression: 87% of YKL-40-positive cases and 86% c-Met-positive cases had a TRG2-5. The overexpression of YKL-40 and c-Met in the non-responder group (TRG2-5) compared with the responder group (TRG1) was statistically significant, when considered both continuous .To verify whether YKL-40 and c-Met co-expression could correlate with a partial or lack of response to neoadjuvant treatment, all cases were stratified into three subgroups, according to the expression of YKL-40 and c-Met on tumor biopsy: i: YKL-40-negative/c-Met-negative , ii: YKL-40-positive/c-Met-negative or YKL-40-negative/c-Met-positive  and iii: YKL-40-positive/c-Met-positive  . This finding strengthens their positive predictive value (94%).When cases were stratified as TRG1-2 vs. TRG3-5 the two biomarkers retained their predictive value, although lower. In detail, 68% of YKL-40-positive cases (p = 0.03) and 65% of c-Met-positive cases (p = 0.01) had a TRG3-5. Stratifying by TRG3, only c-Met retained its value (p = 0.03).Multivariate analysis confirmed YKL-40 and c-Met as independent predictive factors of poor response to CRT .Table 1). In addition, 11 patients (14%) experienced a local recurrence with a median follow-up of 16 months.Follow-up data, ranging from 6 to 79 months (median: 40), were available for all but three patients. At the time of analysis, 75 (96%) of the patients were alive, but the remaining three (5%) had died. Overall, recurrent disease after neoadjuvant CRT and surgery occurred in 21 patients (28%) (All 20 patients identified as complete responders (TRG1) were alive at the time of last follow-up (mean follow-up 52 months), as were 15 of the 16 patients classified as TRG2 (mean follow-up 33 months); one patient in the TRG2 group was lost. The three deceased patients had incomplete tumor response upon surgical rectal specimen , followed by tumor recurrence. Univariate survival analysis showed that high TRG was the only parameter significantly correlated with a shorter overall survival (p = 0.01) and disease-free survival (p = 0.004).In this study, we found that YKL-40 and c-Met immunohistochemical expression in pre-treatment rectal cancer biopsies reliably predicts a partial or lack of response to neoadjuvant CRT in patients with locally advanced rectal adenocarcinoma. Moreover, we demonstrated that c-Met and YKL-40 are independent predictors of poor response to CRT and that Mandard TRG is the only prognostic factor of poor survival.(p = 0.01 and p = 0.006). Interestingly, this observation was further reinforced when only co-expression was considered: 94% of the rectal biopsies co-expressing both biomarkers showed TRG2-5.To date, neoadjuvant CRT is the standard of care for patients with locally advanced (stages II and III) rectal adenocarcinoma. However, its efficacy is quite unpredictable, with a complete pathological response (ypT0) in up to 30% of cases,45,46. SStratifying patients according to TRG2, both YKL-40 and c-Met retained their predictive value (p = 0.03 and p = 0.01 for YKL-40 and c-Met respectively), whereas stratifying by TRG3 c-Met only proved to predict therapy efficacy (p = 0.03).Such a result is in line with the ‘stress-response’ role of the two molecules: tumors highly expressing c-Met and YKL-40 will most likely not benefit from CRT because these cancer cells have already switched on a transcriptional adaptation to unfavorable microenviromental conditions. The decision to use an immunohistochemical approach instead of a molecular analysis provides practical advantages. Genomic and proteomic analyses are expensive, and special equipment and highly trained technicians are required to perform certain molecular analyses, whereas immunohistochemistry is widely employed in diagnostic routines and has a favorable cost-benefit balance. Based on the previous external validation of these multi-institutional results, the introduction of an immunohistochemistry-based panel composed of YKL-40/c-Met in rectal cancer diagnostic-routine in parallel to H&E-stained slides could be used to avoid excessive costs even in laboratories managing a high number of surgical specimens.The role of c-Met in prognosis has been reported to correlate with its overexpression in different solid tumors (such as gastric adenocarcinoma, ovarianYKL-40 and c-Met expression in pre-neoadjuvant rectal cancer biopsy is a reliable tool to predict the partial or lack of tumor response to CRT. Accordingly, an “immunohistochemistry-enriched” diagnostic approach to rectal biopsies, aimed at increasing CRT sensitivity using specifically selected targeted therapies for those expected to be poor responders, could provide predictive information with cost-benefit efficacy and facilitate the further therapeutic selection of patients.Finally, prospective randomized trials are necessary to validate the role of YKL-40/c-Met in predicting tumor resistance; if so, the efficacy of neoadjuvant CRT in these patients would benefit of therapy sensitizers, i.e. targeted c-met inhibitors, to improve responsiveness.


Regression analysis revealed that H-Met was 55.00, 71.00, 78.00, 47.00, 58.00 and 73.00% as efficacious as DL-Met for body weight gain, feed intake, feed conversion ratio, albumin, globulin and high density lipoprotein criteria, respectively. The average of bio-efficacy of H-Met compared to DL-Met was 67.00% and 59.00% on average across performance criteria and blood criteria respectively and was 63.00% across these two criteria tested. The results of the present study indicated that H-Met can be administered as a new and a natural source of Met in poultry industry.This study was conducted to compare the bio-efficacy of herbal methionine (H-Met) relative to DL-methionine (DL-Met) on 160 “Ross 308” broiler chickens. DL-Met and H-Met were added to the basal diet in eight experimental treatments with three and four concentrations respectively in starter, grower and finisher period. Blood parameters which were measured at 24 and 42 days of age consisted of: serum proteins , serum uric acid, serum fats  and serum enzymes . Completely randomized design, multi-exponential and multilinear regressions were used to determine bio-efficacy of H-Met in terms of performance and blood parameters of broilers. The results showed that supplemented methionine (Met) sources had no significant effect on blood parameters at 24 day of age. At 42 day of age the amounts of globulin and serum high density lipoprotein (HDL) increased with supplemented Met, ( Since rapid growth of broilers demands a high quality diet to sufficiently meet their nutrients requirements, it is necessary to supplement these diets with a Met source.2Amino acids are considered the building blocks of proteins. Synthetic amino acids are widely used to enhance protein synthesis in animal by adding them into their diets.3 Increasing prices for petrol-derived precursors of acrolein and methyl mercaptan coupled with the increasing demand for a source of organic Met have urged producers to produce an organic source of Met called H-Met. The most common source of Met in poultry diets is DL-Met. This source of Met is produced by chemical synthesis from acrolein, methyl mercaptan and hydrogen cyanide.4 There are also many studies which have compared the bio-availability of methionine hydroxyl analog-free acid (MHA-FA) relative to DL-Met in broiler chickens.5,Thus, it is necessary to compare this new source of Met with DL-Met in poultry nutrition. Halder and Roy examined the effect of one kind of H-Met as a source of herbal Met and DL-Met on performance of broilers and demonstrated that HerboMet can be used more efficiently than DL-Met.Three and four graded levels of DL-Met (98.00% purity) and H-Met  were added to commercial corn-soybean meal basal diets  and serum was separated and then stored at –20 ˚C until further analysis. Serum samples were analyzed for total protein (TP), albumin (ALB), globulin (GLO), alanine aminotransferase (ALT), aspartate aminotransferase (AST), uric acid (UA), high density lipoprotein (HDL), low density lipoprotein (LDL), total cholesterol (CHOL) and triglyceride (TG). Analysis of blood sample. The concentration of TP was measured by Biuret method and ALB by the bromocresol green method; serum GLO was determined by subtracting serum ALB from TP value. The UA was measured by uricase method, CHOL by the cholesterol esterase-peroxidase method, HDL and LDL cholesterol, and TG using the kit package  and the activities of AST and ALT were determined using automatic analyzer according to the recommendation of the manufacturer.7Statisticalanalysis. Data were evaluated as completely randomized designs and differences between treatment means were tested using Duncan multiple comparison test. The statistical significance was declared at a probability of p < 0.05. The pen mean was considered the experimental unit for all statistical analyses. A nonlinear exponential model was used to estimate the bio-efficacy of H-Met relative to DL-Met as suggested by Littell et al.(c1× x1+ c2× x2))y= a+ b × , b = asymptotic response, a + b = common asymptote (maximum performance level), cet al.8 using the following equation:The FI value was analyzed by multi-linear regression as suggested by Littell 1x1+ b2x2)y = a + . The maximum BWG was achieved by broilers consuming the dietary treatments containing 0.11% DL-Met and 0.17% H-Met (treatments DL-Met-2 and H-Met-3). The improvement in BWG shows that the basal diet was deficient in Met (p < 0.05). The FCR increased by addition of Met sources (p < 0.05). The result of the present study showed that by increasing the level of the Met sources up to 0.11% for DL-Met and 0.17% for H-Met, BWG and FI increased, but the treatments DL-Met-3 (DL-Met at 0.17%) and H-Met-4 (H-Met at 0.22%) fed broilers consumed more feed but less BWG than in treatment DL-Met-2 (DL-Met at 0.11%) and H-Met-3 (H-Met at 0.17%), resulting in increased FCR.t in Met . Feed inSerum metabolites. The effects of dietary treatments on blood metabolites are shown in p < 0.05); and the levels of ALB decreased (p < 0.05). The effect of dietary treatment on TP, UA, LDL, TG, CHOL, AST and ALT were not significant at day 42. 9 It can be assumed that, under amino acid imbalances, chicks lose the potential to adjust FI to satisfy their amino acid requirements; the main positive effect of Met supplementation may come from its improvement of FI via the amino acid balance.11 Theories representing the reasons are: 1) The depletion of hepatic ATP in adenylating Met to S-adenosyl Met (SAM);12 2) Depletion of methyl acceptors in the conversion of SAM to S-adenosyl-homocysteine;13 3) Metabolism of the labile methyl group of Met via an alternate pathway not requiring formation of SAM is the means by which the toxicity is exerted.14 The normal trans-sulfuration pathway of Met catabolism involves formation of homocysteine (HCY), which donates its sulfur moiety to serine to eventually form 1 mole of cysteine per mole of Met catabolized.15 The result of growth performance in the current study did not confirm the result of Halder and Roy who reported that there are no significant differences with utilization H-Met in comparison with DL-Met in the same level.et al. who reported an increase and a subsequent decrease in BWG as dietary Met increased.16 Therefore, it seems that supplemented Met sources more than the required amount for broilers do not improve the BWG. Han and Baker indicated that 0.50% excess of Met is not harmful to young broiler chicks fed corn-soybean meal diet.15 As Met supplementation levels increased regardless of the sources, FI was significantly increased and FCR was also increased due to the higher FI in higher Met supplemented diets.Bunchasak and Keawarun found that Met deﬁciencies depressed the FI of broiler chicks due to amino acid imbalances.17 Feeding low protein and amino acid diets seemed to be associated with a decrease in serum TP and serum ALB in chickens.18 ALB is a blood transport protein which binds many biomolecules and drugs including hormones, lipoproteins and amino acids.19,20Broilers with higher body weight gains showed a higher concentration of serum total protein compared to the lighter broilers possibly due to higher demand for lean tissue maintenance and turnover.,22 One of the reasons for ALB reduction in the current study can be related to its decomposition for supplying amino acids requirements or increasing GLO level. In contrast, Hind et al. showed that the level of Met did not have any effect on serum TP, ALB and GLO, though a numerical increase was observed in the amount of GLO.23Smith suggested that ALB as a major protein in the blood of laying hens is decomposed with an increase in the requirement of amino acids leading to a decrease in its concentration.2124 The results showed that serum HDL increased by addition of Met sources to the diets. Met-supplementation increased HDL and apolipoprotein A-I (apo A-I) in blood.25 Moreover, hepatic mRNA levels and transcription rates of apo A-I gene increased by the addition of Met to soy protein.25 Therefore, the stimulation of apo A-I gene by Met might be responsible for increasing HDL resulted from the addition of Met to the diet.28 It seems that HDL is elevated by sulfur amino acids (SAAs) through an increase in apo A-I gene expression in the liver.26 Taurine, one of SAAs, is synthesized mainly in the liver as an end product of SAAs catabolism and HDL tends to be elevated by dietary taurine.27 Taurine exerts its effect through post-translational modiﬁcation of regulatory proteins such as those associated with phosphorylation/ dephosphorylation or ligand binding to nuclear receptors.26 Reportedly, Met could cause alteration in lipogenesis and lipolysis in broiler chicks.28 This finding indicates that supplementation of Met facilitates efficient lipid metabolism in the liver and its transportation to the tissues and consequently it may reduce the incidence of fatty liver in birds as Halder and Roy reported for Herbo-Met.4Among the amino acids, total sulfur amino acids (TSAA) have the highest potential for adjustment of fat metabolism.et al. demonstrated that the design of the trial can be done either equimolar or weight to weight comparison of the two Met sources, although the results are not exactly the same but both of these methods are true.29 In the present study the addition of each Met sources was done on a weight basis.Hoehler et al. and Payne et al. explained in their studies.5,6 The poor utilization of the polymeric forms of H-Met relative to DL-Met polymeric form may be one of the main reasons for its lower bio-efficacy. There are several hypotheses regarding why H-Met has a lower bio-efficacy relative to DL-Met. There are some possible explanations for lower bio-efficacy of H-Met relative to DL-Met as Hoehler Considering the results of the present study, it seems that H-Met is a suitable natural substitute for DL-Met on the broilers diet, if the cost is suitable. As the results showed, the level of 0.17% from H-Met did not have a significant effect with the level of 0.11% from DL-Met and these two levels of Met sources could obtain the maximum BWG. Therefore, it can be calculated that H-Met should consume 1.55 more times than DL-Met to obtain the same response. Hence, the cost of H-Met should be 45.00% of that of DL-Met in order to be economical. In conclusion, on average bio-efficacy was 63.00% for H-Met based on growth performance and blood parameters. Although, the relative effectiveness of H-Met was significantly lower than DL-Met in broiler chickens, H-Met can be administered as a new and a natural source of Met in poultry industry. Also, as it is stated in above paragraph the cost of H-Met should be considered 45.00% of DL-Met cost to be economical.


IgG4-related disease (IgG4-RD) encompasses a group of fibroinflammatory conditions recognized in recent times. The main clinical features include variable degrees of tissue fibrosis, tumorlike expansions, perivascular lymphocytic infiltration rich in IgG4-positive plasma cells, and elevated serum IgG4. A case has been reported of an elderly patient with an unexplained unilateral exophthalmia; biopsy was performed and revealed lymphocytic infiltration, suggesting IgG4-RD. High serum levels of IgG4, in association with a good response to steroid therapy and to the exclusion of other diagnoses, confirmed the hypothesis of orbital pseudotumor by IgG4-RD. IgG4-related disease (IgG4-RD) is a recently recognized group of fibroinflammatory conditions first described in Japan in 2001. Until now, fewer than 3,500 cases have been reported worldwide, 75% from Asia, and considered very rare in Latin America [The main clinical features include variable degrees of tissue fibrosis, tumorlike expansions, perivascular lymphocytic infiltration rich in IgG4-positive plasma cells, and elevated serum IgG4 . DiagnosWe report a case of an orbital pseudotumor as an unusual first manifestation of IgG4-RD.A 64-year-old white female presented to the outpatient clinic with a 3-year history of left ocular protrusion, associated with pruritus, burning sensation, and excessive tearing. She had no previous medical conditions.On examination, there was left exophthalmo, with unilateral palpebral oedema, conjunctival hyperemia, and pain resulting from ocular globe compression. Ocular motricity was preserved, but visual acuity was reduced on the right field, with blurred vision on the right. The remainder of the physical examination was unremarkable.Laboratory tests of thyroid function and autoantibodies were normal. Kidney function was appropriate for age, and pancreatic and hepatic enzymes were in the normal range. Search for antinuclear antibodies (ANA), anti-SSA/Ro, and ANCA showed negative results, and viral serologies  were negative. Immunoglobulin G class 4 was 829 mg/dL .A computed tomography (CT) scan of the head showed expansive lesion in the left orbita, with a solid mass on the retro-orbital space and contrast enhancement of rectus medial and rectus lateral of the corresponding eye . kappa and lambda, with polyclonal aspect.A biopsy was done  and reveAs the proptosis was increasing, it was decided to start on prednisone 1 mg/Kg/day for eight weeks, followed by azathioprine 2.5 mg/Kg daily. After six months of treatment, another CT scan showed reduction in the protrusion and; after one year, the patient recovered her visual acuity and had almost no exophthalmo Figures .IgG4-RD is a recently recognized syndrome with many unexplained aspects, but with specific serological, clinical, and histological features. It generally presents with a unique organ infiltration and is frequently misdiagnosed as malignancy . Many reThe first case reported was described by Hamano et al.: several young patients with pancreatitis with no clear etiology and high serum levels of IgG4 ended up developing pancreas sclerosis . Since tClinical aspects are variable and are more often described in old males in their sixth decade of life: sclerosing cholangitis, glomerulopathies, dacryoadenitis and sclerosing sialadenitis, idiopathic fibrosis , pulmonary inflammatory pseudotumors and interstitial pneumonitis, pachymeningitis, and hypophysitis –7. The pOn complementary methods, serum levels of IgG4 higher than 135 mg/dL used to be a diagnostic criterion for IgG4-RD. However, initial stages of the disease, or a patient with immunodeficiency, may present with normal levels of the biomarker, in which it is preferable to use the relation of serum IgG4/total IgG higher than 8% as a high sensitive and specific criterion . HistopaLacrimal and orbital involvements are rare and are called IgG4-related ophthalmic disease, frequently presenting as orbital myositis, perineuritis of the optic and trigeminal nerves, orbital inflammation, or scleritis . Most coSince the first clinical feature presented in this case was orbital pseudotumor, the most common differential diagnosis had to be excluded. As the thyroid exam was normal and the extraocular muscles were preserved on the CT scan (in accordance with what is expected in IgG4-RD), exophthalmos due to Graves' disease was less probable . SjögrenGlucocorticoids are the first-line therapy to IgG4-RD, regardless of the organ involvement , 4, 6, 9Azathioprine, methotrexate, and mycophenolate are used with limited success in patients with advanced IgG4-RD and corticosteroid dependency and, in these cases, the use of rituximab may be beneficial . In caseRecently, an association of B cell lymphoma with IgG4-RD  has beenThe relevance in reporting IgG4-RD cases, especially in unusual scenarios, such as the orbital pseudotumor, remains an important contribution to the many possible disease presentations, as this is still an underdiagnosed condition in which the treatment may lead to remission and prevent significant morbidity and mortality. Future studies should focus on identifying biomarkers to assist with noninvasive methods of diagnosis and testing novel treatments that might supplant glucocorticoids as first line agents of treatment, providing better responses or, at least, requiring minimal doses of steroids. We believe that anti-B cells therapies (such as rituximab or belimumab) may be promising choices, as the disease probably relies on B-lymphocytes to progress.


In this study, we defined a new parameter, referred to as the cardiac stress index (CSI), using a nonlinear detrended fluctuation analysis (DFA) of heart rate (HR). Our study aimed to incorporate the CSI into a cycling based fatigue monitoring system developed in our previous work so the muscle fatigue and cardiac stress can be both continuously and quantitatively assessed for subjects undergoing the cycling exercise. By collecting electrocardiogram (ECG) signals, the DFA scaling exponent α was evaluated on the RR time series extracted from a windowed ECG segment. We then obtained the running estimate of α by shifting a one-minute window by a step of 20 seconds so the CSI, defined as the percentage of all the less-than-one α values, can be synchronously updated every 20 seconds. Since the rating of perceived exertion (RPE) scale is considered as a convenient index which is commonly used to monitor subjective perceived exercise intensity, we then related the Borg RPE scale value to the CSI in order to investigate and quantitatively characterize the relationship between exercise-induced fatigue and cardiac stress. Twenty-two young healthy participants were recruited in our study. Each participant was asked to maintain a fixed pedaling speed at a constant load during the cycling exercise. Experimental results showed that a decrease in DFA scaling exponent α or an increase in CSI was observed during the exercise. In addition, the Borg RPE scale and CSI were positively correlated, suggesting that the factors due to cardiac stress might also contribute to fatigue state during physical exercise. Since the CSI can effectively quantify the cardiac stress status during physical exercise, our system may be used in sports medicine, or used by cardiologists who carried out stress tests for monitoring heart condition in patients with heart diseases. Fatigue is often considered as an important indicator in diseases due to abnormality or a progressive decline in motor function during motor tasks –7. In faIn fact, fatigue can be classified as central and peripheral fatigue; the former is associated with the central nervous system (CNS) while the latter is associated with the peripheral neuromuscular system –14]. Th. Th14]. Considering the local muscle factors first, a reduction in median frequency of the EMG power spectrum has been widely accepted as an indicator of fatigue since it has been observed in fatigue induced by maximum and sub-maximum voluntary contractions –19. In fHowever, one of the interesting questions usually arising from endurance training is often asked: “Is the one who underwent such a training test truly fatigued or just lacking in motivation?” Seeking for the answers to the question actually motivated this study. Although heart rate (HR) would increase when one exercises, an increase in HR does not necessarily indicate fatigue. On the other hand, fatigue induced by physical exercise may be partly attributed to an increase in cardiac stress. In fact, the idea of a relationship between cardiac stress and fatigue is not original. Since intensive exercise will be obviously related to incremented sympathetic tone and reduced parasympathetic tone, heart rate variability (HRV) analysis may provide a more effective and reliable way for figuring out this relationship. In this aspect, some previous researches in literature, for instance, have reported that HRV may and does show influence of mental and physical stress –28]..28].It has been widely accepted that the autonomic nervous system (ANS) plays a direct role in the ruling action of the HR control. The CNS in brain receives various stimuli and responds to these stimuli via the ANS activity. The ANS activity modulates the heart rate as well as other cardiovascular variables, such as blood pressure, and then feeds back to the CNS again. Since such a control is close-loop and running continuously, the HRV analysis thus provides a noninvasive way for the study of ANS modulation. There are a number of previous studies in the literature having indicated that HRV analysis may provide an insight into the mechanism of ANS activity –31. In gα during the graded cycling exercise test, all their data processes were done after all the RR data were collected, i.e. not real-time. In this study, we defined an innovative time-varying parameter based on the DFA scaling exponent, dubbed the cardiac stress index (CSI), and incorporated it into the cycling based fatigue monitoring system developed by us previously so the muscle fatigue and cardiac stress can be synchronously monitored during the physical exercise. We believe such a novel system can be used in sports medicine, or used by cardiac clinicians for on-line monitoring heart condition in patients with heart diseases when undergoing stress tests.As mentioned in the very beginning, we have developed a cycling movement based system for on-line fatigue monitoring and analysis in a previous work. In this study, we further examined the effects of cycling exercise on HR dynamics at a constant load. The hypothesis of our study was partly based on an extension of another previous work in literature regarding the DFA with its applications into heart rate dynamics on aerobic cycling exercise tests . AlthougThe study protocol had been approved by Chang Gung Medical Foundation Institutional Review Board (IRB no.101-5141B) in accordance with the Helsinki Declaration. All participants have provided their written consent, approved by Chang Gung Medical Foundation Institutional Review Board (IRB no.101-5141B), to participate in this study.In order to perform rhythmic movement trainings, a stationary bicycle based ergometer system was developed and used. The information regarding our cycling-based ergometer system is as depicted in e.g., FPM and CSI) used for on-line fatigue and cardiac stress monitoring and analysis.We here adopted EMG and ECG sensors to collect medical data from the subjects. The software programs were devised to process these medical signals to extract the diagnostically useful information in an integrated fashion. In summary, the system PC was used to carry out the on-line analyses as follows: 1) acquiring and displaying the ECG, EMG, HR, and cycling speed data, 2) preprocessing the collected raw ECG and EMG data, 3) computing the fast Fourier transform (FFT) based power spectrum and the DFA scaling exponent, 4) transforming all the numerical results into fatigue and cardiac stress related parameters  and gastrocnemius (GAS) muscles, ECG, instantaneous HR, cycling velocity and cycling time were all synchronously recorded.In addition, it has been known that the Borg rating of perceived exertion (RPE) scale is used to reflect how hard a participant feels about the work when exercising . With raIn addition to the EMG-based fatigue analysis, in this study the detrended fluctuation analysis (DFA) was further introduced to the characterization of HR dynamics. For this purpose, the subject’s surface ECG was measured and digitized by an ECG sensor interfacing system first. The digital ECG data were then wirelessly transmitted to the system PC through Bluetooth. Note that here the sampling rate was set to 200Hz and the raw ECG data was preprocessed by a bandpass filter. After detecting the time locations of the R waves for an ECG signal using a template-match technique that involves a correlation coefficient based similarity calculation, we may obtain the interbeat interval  data sequence corresponding to that ECG. On the other hand, it should be also noted that for online or mobile HRV analysis ectopic beats would generally cause abnormal RR intervals, or alternatively known as the outliers, that occur randomly and are longer or shorter than normal RR data. This would represent a major source of error when analyzing HRV data in both the time and frequency domain. Therefore, in order to solve this problem, an algorithm used for detecting and editing RR outliers was applied before performing DFA. In the editing process, we deleted the RR outliers detected and then replaced the deleted ones simply by linearly interpolated RR values. In fact, some previous researches in literature reported that the effects of editing in DFA calculation may not be significant when the number of outliers is small  –39. In oet al. . I. Iet al. classes . In geneF(n) and the time scale n , the calculation in (2) is repeated until all possible values of n are applied. In general, a linear relationship is seen on a log-log plot and the fluctuations can be then characterized by the slope of the line relating log F(n) to log n. Also note that in our study, n ranged from 4 to 64.In order to characterize the relationship between α derived from healthy and unhealthy  subjects, respectively . Mo. Moα ders status –33]. Th. Thα derα counts. After cycling exercise starts, the denominator in (3) will be incremented by one every time when there is a new α value produced, while the numerator is incremented by one only when α is lower than 1. According to the definition in (3), the CSI can be used as a diagnostically useful indicator to continuously and quantitatively describe the progression of cardiac stress status as time evolves.Similar to the way that FPM is defined (as described in detail previously ), (3) isRecall in our previous study, we have devised another time-varying parameter, dubbed fatigue progression measure (FPM), based on the percentage of reduced median frequencies (MFs) of the EMG spectrum for quantitatively and continuously measuring the degree of fatigue . Accordind number, the FPM sequence would be 0/1, 1/2, 2/3, 3/4, 4/5,…, 9/10; if crossed at the 3rd point, it would be 0/1, 0/2, 1/3, 2/4, 3/5, 4/6, … 8/10. Obviously, FPM value will remain at zero until there is a MF number crossing  the threshold. In fact, in our latest system, as presented in this study, the α value and the filtered MF value were both synchronously produced every 20 seconds and thus, the time-varying parameters, FPM and CSI, were updated every 20 seconds to provide the continuous measures of fatigue and cardiac stress status, respectively. Figs α tracings, CSI and FPM tracings, respectively.Here, we simply set the reference  as first MF value obtained from the cycling test. Note that since MF is updated as time evolves, the FPM  would be synchronously updated. For example, given 10 MF numbers, when the threshold is crossed at the 2α throughout the entire cycling test can be derived, as depicted in α (tk) by shifting a 1-minute analysis window centered at tk by a step of 20 seconds on the RR data. Observing α was seen. This might be regarded as the result of an increase in cardiac stress [α signal were further evaluated and updated by successively comparing the α values with the reference value, 1.0, using (3) and plotted in c stress . The CSIAs mentioned previously, we collected the raw EMG as well as ECG data from twenty-two subjects during the cycling tests in order to evaluate the overall performance of our system with its applications into real-time muscle fatigue and cardiac stress assessment. Note that among all the physiological measures employed in our system, the CSI method as described in previous subsection is innovative and new. Being similar to the FPM with respect to continuous muscle fatigue monitoring, the CSI can not only detect the onset time at which the cardiac endurance starts to decrease, but also continuously measure the progression of cardiac stress. Therefore, we believe this would represent the most significant benefit from this work. The subsequent numerical analysis results and discussions will be focused on the performance evaluation of both the FPM and CSI methods.α, and mean CSI, respectively, of all the twenty-two subjects evaluated at the four stages in the course of cycling exercise. It is revealed from these figures that while the mean HR of these subjects increased from 143 beats/min to 170 beats/min during the cycling exercise, the mean DFA scaling exponent measure α decreased from 1.07 to 0.69, implying that the HR became less regular and/or more complex as the cycling time increased and thus the CSI increased from 0.26 to 0.70 , as expected.Note that in our study, a constant load was applied for all the twenty-two subjects. Evaluation on CSI stopped at the time when the subject’s Borg RPE scale value reached 20. First, it should be noted that since there was considerable diversity in the cycling time of all the subjects undergoing the tests, in order to facilitate the task of performance evaluation we here evenly divided the cycling time interval of each subject into four sections/stages so the stochastic analysis may be conveniently performed. Figs Moreover, we also depicted the mean FPM plots derived from the twenty-two subjects’ VL and GAS muscles in Figs CFPM_VL, CFPM_GAS, CCSI and C0 are unknown constants called the modeling parameters. Note that the coefficients CFPM_VL, CFPM_GAS, CCSI control the behavior of the model and can be used to quantify the attribute and strength of the relationship between Borg RPE scale and FPM of VL and GAS muscles, and CSI, respectively. Moreover, a quantity referred to as the coefficient of determination R2, ranging from 0 to 1, is used to measure the proportion of the dependent variable, i.e., Borg scale, attributable to the information obtained from the independent variables, FPM_VL, FPM_GAS, and CSI, in the multivariate linear model.In general, the main purpose of the linear regression analysis here is to use mathematical expressions to describe the Borg RPE scale in terms of the FPM and/or CSI. The scenario of our numerical experiment is described as follows. First, we let the Borg RPE be the dependent variable, and let both the FPM and CSI be independent variables. That is, we haveCFPM_GAS were positive, indicating that the FPM of VL muscle and CSI were positively correlated with the Borg value. It should be also noted that although the mean of CFPM_GAS was negative, it may not be able to reach a satisfactory significance level (since p = 0.259). Moreover, the averaged coefficient of determination R2  was 0.86, indicating that 86% of the variation in the Borg RPE scale can be explained by a joint analysis of FPM and CSI in the linear model.As described previously, FPM of VL and GAS, as well as CSI were evaluated and updated as time evolved. Also, the Borg RPE scale value was synchronously recorded during the cycling test. Therefore, we first performed a multivariate linear regression analysis, as indicated in (5), on the Borg scale, FPM and CSI data derived from each subject and tabulated all the statistical analysis results in Secondly, we related the Borg scale to the FPM data only using the linear regression model as follows:CFPM_VL and CFPM_GAS were both positive, indicating that the FPM of VL and GAS muscles were both positively correlated with the Borg value, as expected. Also, the mean value of R2 was only 0.68, indicating that only 68% of the variation in the Borg RPE scale can be explained by FPM of VL and GAS in the linear model.Similarly, we tabulated all the statistical results in CCSI was positive with p<0.01 (p = 0.0007), suggesting that the CSI was significantly positively correlated with the Borg RPE value. In addition, the mean value of R2 was also 0.68 in this case, evidently indicating that there was 68% of the variation in the Borg RPE scale, at most, attributable to the non-motor features of fatigue due to cardiac stress in the linear model.Finally, we quantified the association between the Borg scale level and the cardiac stress by fitting the Borg scale and CSI data to the following linear regression modelFirst, it should be noted that analysis results produced by numerical experiments as presented above have substantially validated the use of CSI in cardiac stress quantification. Secondly, just as the FPM, a typical CSI curve also shows an exponential-like increase as time evolves after the time instant at which the cardiac stress starts to increase (or the cardiac endurance starts to decrease). Further observing and comparing results presented in R2 value, the model in (5) achieved 86%, indicating that the Borg scale may be attributable to both the local muscle fatigue and cardiac stress. Secondly, considering the model in (5), we may see from C0 = 10.68 actually implied that either the muscle fatigue or cardiac stress might start to contribute to Borg scale increment from Borg = 11.Moreover, it has been widely accepted that the Borg RPE scale can be used to rate the degree of perceived intensity of exercise . In our R2 was only 68%, manifesting that in addition to local muscle fatigue, the Borg scale should be also attributable to the factors other than muscle fatigue. Secondly, we found that the effects on Borg scale due to FPMs of VL and GAS muscles, respectively, were almost comparable. Thirdly, another interesting note is in this case the mean intercept C0 was 13.37, which indicated that the muscle fatigue might start to contribute to Borg scale increment from Borg = 13  or so. That is, this might imply that the onset of muscle fatigue generally occurred when subjects rated 13 on Borg scale. It is worth noting that such an observation is actually consistent to that found in our previous work [Furthermore, consider the case of relating the Borg scale to FPM only, as indicated by the linear model in (6). First, we may see from the analysis results as provided in ous work .R2 was only 68%, both the statistical results of CCSI and C0 achieved the highest significant level (with p<0.01), in comparison to the numerical results as tabulated in Tables C0 was 10.23, which was actually consistent to the results in Similarly, we also attempted to relate the Borg scale solely to CSI using the linear model as expressed in (7) and obtained the results as shown in In summary, the numerical experimental results produced by a nonlinear analysis of HRV indicated that the cycling exercise effects on HR dynamics can be continuously and quantitatively captured by CSI. Moreover, we further incorporated the CSI into our system so the onset of the occurrence of non-motor features of fatigue due to cardiac stress “overload” can be explicitly determined from the CSI tracings. In the authors’ opinion, in addition to providing an on-line muscle fatigue monitoring and analysis, the proposed system may be also used in sports medicine, or used by cardiologists who carried out stress tests for monitoring heart condition in patients with heart diseases, thus representing a significant benefit from this study.In this paper, a cycling-based training system for real-time muscle fatigue and cardiac stress monitoring and analysis is introduced. In fact, RPE scale has been widely accepted as a convenient index which is often used to determine the level of fatigue during physical exercise. However, since fatigue induced by exercise is a complex phenomenon which may not be solely related to RPE, our study thus aimed to develop novel indices, other than RPE, and seek for their applicability for objectively and quantitatively monitoring and assessing muscle fatigue and cardiac stress during physical exercise. A new parameter, dubbed the CSI, used to quantify the cardiac stress status of subjects undergoing cycling exercise was developed. Similar to how the FPM was used to monitor the progression of muscle fatigue, the CSI was also incorporated into the system that was developed in our previous work for continuously and quantitatively assessing the progression of cardiac stress during exercise. In addition, in order to validate the applicability of FPM and CSI into exercise-induced fatigue analysis, we further related the Borg RPE to both the FPM and CSI using a multivariate linear regression analysis. Numerical experimental results indicated that the Borg RPE was generally positively correlated with FPM and/or CSI, suggesting that the motor factors and the factors due to cardiac stress might both contribute to fatigue state during physical exercise.


Critical Care, Lorente and colleagues reported the results of a prospective observational study aiming at evaluating the effect of continuous control of cuff pressure (Pcuff ) on the incidence of ventilator-associated pneumonia (VAP). The results suggest a beneficial impact of this intervention on VAP prevention, which is in line with the results of a recent randomized controlled study. However, another randomized controlled study found no significant impact of continuous control of Pcuff on VAP incidence. Several differences regarding the device used to control Pcuff, study population, and design might explain the different reported results. Future randomized multicenter studies are needed to confirm the beneficial effect of continuous control of Pcuff on VAP incidence. Furthermore, the efficiency and cost-effectiveness of different available devices should be compared. Meanwhile, given the single-center design and the limitations of the available studies, no strong recommendation can be made regarding continuous control of Pcuff as a preventive measure of VAP.In a previous issue of  Critical Care, Lorente and colleagues [cuff) on the incidence of ventilator-associated pneumonia (VAP). They included a large number of patients intubated and ventilated for more than 48 hours (150 patients in a continuous control group and 134 patients in a routine care group). The target Pcuff was 25 cm H2O in both groups. Whereas the main clinical characteristics were similar in the study groups, VAP rate was significantly reduced by the use of continuous control of Pcuff. Additionally, continuous control of Pcuff and use of subglottic secretion drainage were independently associated with reduced incidence of VAP. Kaplan-Meier analysis showed a significantly higher proportion of patients remaining free from VAP using continuous control of Pcuff compared with intermittent control using a manometer.In a previous issue of lleagues  reportedcuff on tracheal ischemic lesions was not evaluated. However, previous animal and clinical studies found no significant effect of this intervention on the incidence of intubation-related tracheal damage [cuff was not previously evaluated in critically ill patients.The strengths of this study are the large number of included patients and adjustment for confounders using Cox proportional analysis. As acknowledged by the authors, the absence of randomization and blinding is one of the limitations of the study. In addition, the impact of continuous control of Pl damage ,3. Furthcuff is in line with the results of a recent randomized controlled study performed by our group [cuff on VAP incidence [cuff, study population, and design might explain the different reported results  of the tracheal cuff. In a cohort of 101 critically ill patients intubated with a polyvinylchloride-cuffed tube, Pcuff was continuously recorded for 8 hours after manual adjustment of Pcuff at 25 cm H2O [2O) Pcuff. Fifty-four percent of study patients developed cuff underinflation, 73 % developed cuff overinflation, and 44 % developed both. Subsequent studies reported similar results in patients intubated with polyurethane-cuffed tracheal tubes [cuff, including the one used by Lorente and colleagues, are currently available on the market. Unfortunately, the efficiency of some of these devices was never tested in clinical studies. Therefore, before these devices can be used in critically ill patients, well designed and performed studies are required.In spite of routine manual control of P5 cm H2O . Only 18al tubes ,15. Sevecuff on VAP incidence. Furthermore, the efficiency and cost-effectiveness of different available devices should be compared. Meanwhile, given the single-center design and the limitations of the available studies, no strong recommendation can be made regarding continuous control of Pcuff as a preventive measure of VAP.Future randomized multicenter studies should confirm the beneficial effect of continuous control of P