In high-income settings, the spectrum of morbidity and mortality experienced by Human Immunodeficiency Virus (HIV)-infected individuals receiving combination antiretroviral therapy (cART) has switched from predominantly AIDS-related to non-AIDS-related conditions. In the context of universal access to care, we evaluated whether that shift would apply in Brazil, a middle-income country with universal access to treatment, as compared to France.Two hospital-based cohorts of HIV-infected individuals were used for this analysis: the ANRS CO3 Aquitaine Cohort in South Western France and the Evandro Chagas Research Institute (IPEC) Cohort of the Oswaldo Cruz Foundation in Rio de Janeiro, Brazil. Severe morbid events (AIDS- and non-AIDS-related) were defined as all clinical diagnoses associated with a hospitalization of ≥48 hours. Trends in the incidence rate of events and their determinants were estimated while adjusting for within-subject correlation using generalized estimating equations models with an auto-regressive correlation structure and robust standard errors.Between January 2000 and December 2008, 7812 adult patients were followed for a total of 41,668 person-years (PY) of follow-up. Throughout the study period, 90% of the patients were treated with cART. The annual incidence rate of AIDS and non-AIDS events, and of deaths significantly decreased over the years, from 6.2, 21.1, and 1.9 AIDS, non-AIDS events, and deaths per 100 PY in 2000 to 4.3, 14.9, and 1.5/100 PY in 2008. The annual incidence rates of non-AIDS events surpassed that of AIDS-events during the entire study period. High CD4 cell counts were associated with a lower incidence rate of AIDS and non-AIDS events as well as with lower rates of specific non-AIDS events, such as bacterial, hepatic, viral, neurological, and cardiovascular conditions. Adjusted analysis showed that severe morbidity was associated with lower CD4 counts and higher plasma HIV RNAs but not with setting (IPEC versus Aquitaine).As information on severe morbidities for HIV-infected patients remain scarce, data on hospitalizations are valuable to identify priorities for case management and to improve the quality of life of patients with a chronic disease requiring life-long treatment. Immune restoration is highly effective in reducing AIDS and non-AIDS severe morbid events irrespective of the setting. Currently, individuals infected by the human immunodeficiency virus (HIV) and treated with combination antiretroviral therapy (cART) live longer than ever in high-income countries . The speWhereas causes of death are now well described ,4, severOur aim was to describe and contrast severe morbidity trends and determinants among HIV-infected individuals followed in two geographically contrasting settings (France and Brazil) with universal access to treatment and care. To this end, we studied severe morbidity over a 9-year period in two hospital-based cohorts of HIV-infected individuals: the ANRS C03 Aquitaine (Aquitaine) in South Western France and the Evandro Chagas Research Institute (IPEC) Cohort of the Oswaldo Cruz Foundation in Rio de Janeiro, Brazil.The ANRS CO3 Aquitaine Cohort is an open prospective cohort of HIV-1 infected patients followed in nine public hospitals of the Aquitaine region in Southwestern France. The cohort was initiated in 1987 and includes adults with documented HIV infection regardless of clinical stage and informed consent. Details of the cohort can be found elsewhere . The ANRThe Evandro Chagas Clinical Research Institute (IPEC) has provided care to HIV-infected patients in Rio de Janeiro since 1986. An observational, longitudinal, clinical database has been maintained on patients receiving primary HIV care in the clinic since 1998. Longitudinal data are updated regularly using outpatient and inpatient clinical documentation, laboratory testing results, and pharmacy records. Prescription of cART  is documented by the medical provider and support staff in the clinical records. Trained abstractors record all this information onto standardized forms for processing. Details of the cohort have been described elsewhere . The IPEThe sample selected for this analysis included all adult patients (age ≥ 18 years) followed in each cohort from 2000 to 2008.A severe morbid event was defined as a clinical diagnosis associated with a hospitalization with at least 48 hours of duration. For each hospitalization, all diagnosis listed in the hospital records at hospitalization discharge were counted as separate severe morbid events. That is, one hospitalization could be associated with several clinical diagnoses, and, in this case, each different diagnosis was taken into account. Dates of admission and discharge were captured among both discharged patients and those who died during hospital stay.The ANRS C03 Aquitaine Cohort used a simplified version of the International Classification of Diseases (ICD-10) and the IPEC Cohort used ICD-10 to code the clinical diagnoses. As some codes could correspond to several disease categories, we considered a hierarchical classification with a decreasing order of priority considering AIDS-events, non-AIDS malignancies, infections and systemic events. Signs and syndromes were considered as events if they were not reported along with other diagnoses during a specific hospitalization. Hospitalizations due to a non-morbid cause , whatever the duration of the stay, were not considered in our definition of severe morbidity and were not included in this analysis. All events retrieved from the database were reviewed and validated by two clinicians highly experienced in the management of HIV infection, one clinician from Brazil and another from France. The lists of codes allocated to the different disease categories by each cohort were then compared and all discrepancies were discussed until a consensus was reached.Severe morbid events were classified under 22 disease categories as follows: (1) AIDS, (2) Non-AIDS malignancies, (3) In situ malignancies, (4) Cardiovascular, (5) Hepatic, (6) Digestive, (7) Psychiatric, (8) Hematological, (9) Renal, (10) Endocrine, (11) Bacterial, (12) Viral, (13) Parasitic, (14) Dermatological, (15) Gynecological, (16) Neurological, (17) Ophthalmological, (18) Respiratory, (19) Rheumatologic, (20) Trauma, (21) Signs and syndromes, and (22) Other.3. For consistency across years, 400 copies/ml of plasma HIV RNA was defined as the limit of detection. Plasma HIV RNA was categorized into ≤ 400, 401–3000, 3001–10000, 10001–100000, >100000 copies/ml. Time since first HIV positive test was calculated for each calendar year. cART was defined as two nucleoside reverse transcriptase inhibitors plus either one non-nucleoside reverse transcriptase inhibitor or one protease-inhibitor (boosted or not). Hepatitis B infection was defined by the presence of hepatitis B surface antigen (HBsAg) while hepatitis C infection was defined by the presence of hepatitis C antibodies.Age from birth was updated yearly for each participant. Presumed mode of HIV exposure was classified as men who have sex with men (MSM), injection drug use (IDU), heterosexual transmission, other, and unknown. Yearly CD4 cell counts and plasma HIV RNAs were defined as the mean value of all measurements taken in a specific calendar year. CD4 cell counts were categorized into >500, 500–351, 350–201, 200–51, ≤ 50 cells/mmDescriptive analyses of the demographic and clinical characteristics of the study patients were conducted, including age, gender, mode of HIV transmission, CD4 cell count, plasma HIV RNA, time since first HIV positive test, ever use of cART, and hepatitis B and C co-infections.Each patient contributed to multiple years of observation, one for each calendar year. Patients could enroll in the cohort at any time preceding or during the study period (1 January 2000 to 31 December 2008), and thus the number of person-years was not constant across patients or years. Within each year, we calculated the number of days of follow-up. If a patient enrolled in a given year, the number of days prior to enrolment was excluded from the count of number of days of follow up for that year. Individuals were not censored after an episode of hospitalization, that is, readmissions were also studied, irrespective of the reason for hospitalization. The end of follow-up was defined as the last contact with the clinic, 31 December 2008, or death, whichever occurred first.We estimated the rate of severe morbid events overall and by category for each calendar year using as numerator the number of severe morbid events and as denominator the person-years (PY) of follow-up. To assess trends over time, calendar year (2000 to 2008) was included in the regression model and the significance of the per year increase coefficient was tested. Age, CD4 cell count, plasma HIV RNA, time since first HIV positive test, ever use of cART, and hepatitis B and C co-infection were updated for each year of follow-up. Adjusted incidence rate ratios were used to measure the degree of association of demographic and clinical factors with the overall incidence rate of AIDS, death, and non-AIDS events, as well as the specific incidence rate of non-AIDS events categories.We used generalized estimating equations (GEE) models with an auto-regressive correlation matrix and robust standard errors to adjust for the clustered nature of the data here represented by the multiple observation years for each patient (assumption of independence between clusters). The specified working correlation structure (i.e. the auto-regressive correlation matrix) describes how the responses  within clusters (patients) are related to each other. GEE models have been extensively used for data in which the responses are correlated ,15. The Between January 2000 and December 2008, 7812 patients were followed for a total of 41,668 person-years (PY) of follow-up, 29,866 PY for Aquitaine and 11,802 PY for IPEC. The mean follow-up time per patient for the two cohorts combined was 5.3 years . Approximately two-thirds of the study population was from Aquitaine and its mean follow-up time per patient was 6.0 years , compared to 4.2 years  for IPEC.3 in 2008; in addition two-thirds of the population had undetectable plasma HIV RNA in 2008. The prevalence of hepatitis C decreased over time. Ageing, decreased proportion of IDUs, and improved immunological profile were observed in the two cohorts when evaluated separately, though trends were more pronounced in the Aquitaine Cohort  participants, 1473 (29.5%) from Aquitaine and 765 (27.1%) from IPEC. A total of 4689 hospitalizations were recorded, 3049 (65.0%) in Aquitaine and 1640 (35.0%) in IPEC. One severe morbid event was reported in 2348 hospitalizations (50.1%), two morbid events in 1359 hospitalizations (29.0%), and three in 599 hospitalizations (12.8%). In 383 (8.2%) hospitalizations more than three severe morbid events were reported. Among those who were hospitalized , 8696 severe morbid events were reported, 5963 (68.6%) in Aquitaine and 2733 (31.4%) in IPEC. There were 310 deaths reported, a 6.6% fatality ending hospitalizations.Over the entire study period, the incidence rate of severe morbid events was 21.5/100 PY : 20.3 to 22.8/100 PY) overall, 20.5/100 PY (95% CI 19.1 to 22.0/100 PY) in Aquitaine and 24.0/100 PY (95% CI 21.8 to 26.4/100 PY) in IPEC. The annual incidence rate of severe morbid events significantly decreased from 27.3/100 PY in 2000 to 19.2/100 PY in 2008 . In Aquitaine, 498 patients died during the study period and the mortality rate decreased from 1.6/100 PY in 2000 to 1.1/100PY in 2008 (p < 0.01). In IPEC, 393 deaths occurred yielding a higher mortality rate that also decreased from 3.0/100 PY in 2000 to 2.3/100 PY in 2008 although the difference did not reach statistical significance (p = 0.09).The annual incidence rates of the 21 non-AIDS disease categories are displayed in Table The three most frequent diagnoses within the AIDS category and specific non-AIDS categories are shown in Table Adjusted incidence rate ratios quantifying the degree of association of demographic and clinical factors with the incidence rate of AIDS and non-AIDS events are shown in Table The incidence of non-AIDS events was increased for those with older age and who reported injection drug use. The incidence of non-AIDS events was also significantly higher for those with less than 1 year since first HIV positive test, those who used cART, and those with hepatitis C co-infection. Of note is the fact that, even after adjusting for confounders, IPEC still showed a lower incidence of non-AIDS events when compared to Aquitaine.Age ≥ 60 years was associated with a higher incidence rate of several specific non-AIDS events categories, including bacterial, hepatic, neurological, and cardiovascular. Age ≥ 60 years showed a particularly strong association with cardiovascular disease  with universal access to treatment and care in the cART era. Over a 9-year period (2000–2008), ageing, decreased representation of IDUs, and improved immunological profile were observed in both cohorts largely exposed to cART. The rate of severe morbid events decreased by one-third over the period and, in 2008, only one out of five events was AIDS-related; non-AIDS events were always more frequent than AIDS events. Immunodeficiency was associated with a higher incidence of AIDS-related and all types of non-AIDS-related events. Our observations suggest that long-term restoration and preservation of high CD4 cell counts as a result of cART access is highly and durably effective in preventing AIDS as well as non-AIDS severe morbid events.In this collaboration, sizeable efforts were allocated for the collection, documentation and uniform classification and coding of morbid events leading to hospitalizations. There is indeed a lack of published studies on the long-term causes of severe morbidity leading to hospitalization in HIV-infected patients, although such information is highly valuable in identifying priorities for case management and improvement of the quality of life of subjects living with a chronic disease requiring life-long treatment. Our study population combined two hospital-based cohorts that mainly differed by environment and only modestly by access to care. In this context, non-AIDS related conditions were the most common cause of hospitalizations throughout the study period.3 and undetectable plasma HIV RNAs. In fact, even beyond severe immunodeficiency, individuals with CD4 cell counts between 350–500 cells/mm3 were found to have a three times higher incidence rate of AIDS events than those with CD4 cell counts >500 cells/mm3. In addition, although the comparison of the incidence rate of AIDS events in Aquitaine and IPEC initially suggested that AIDS events occurred at much higher frequency in the latter, after adjustments for other characteristics , this finding did not hold, suggesting that environment is not a primary determinant of the occurrence of AIDS severe diseases in a population highly exposed to effective antiretroviral combinations ) of the IPEC Cohort study population by year. Table S3. Annual incidence rates per 100 person-years of non-AIDS events ranked from most to least frequent for the ANRS CO3Aquitaine Cohort. Table S4. Annual incidence rates per 100 person-years of non-AIDS events ranked from most to least frequent for the IPEC Cohort. Table S5. Three most frequent diagnoses within the AIDS and specific non-AIDS severe morbid events categories in the ANRS CO3 Aquitaine Cohort. Table S6. Three most frequent diagnoses within the AIDS and specific non-AIDS severe morbid events categories in the IPEC Cohort.Demographic and clinic characteristics (number [percent]) of the ANRS CO3 Aquitaine Cohort study population by year. Click here for file


Childhood obesity and overweight are among the greatest health challenges in the pediatric population. Obese individuals exhibit marked differences in the composition of the intestinal microbial community as compared to lean subjects. These changes in the gut microbiota precede the clinical manifestation of overweight. Convincing experimental data suggest a causal role for intestinal microbes in the development of obesity and associated metabolic disorders.Exposure to antibiotics exerts a devastating impact on the intestinal microbial community. Epidemiological studies have provided evidence indicating that early or repeated childhood exposure to antibiotics is associated with increased risk of overweight later in childhood but the causal role of this exposure in obesity development is not clear. However, data from studies conducted using experimental animal models indicate that antibiotic-induced changes in the gut microbiota influence host metabolism and lead to fat accumulation. The intestinal microbiota perturbation caused by antibiotic exposure in the perinatal period appears to program the host to an obesity-prone metabolic phenotype, which persists after the antibiotics have been discontinued and the gut microbiota has recovered. These observations may have serious implications in the clinical setting, since a substantial number of human infants are subjected to antibiotic treatment through the mother during delivery or directly in the immediate neonatal period. The clinical significance of these exposures remains unknown.Prudent use of antibiotics is paramount not only to reduce the propagation of antibiotic-resistant organisms but also to minimize the potentially detrimental long-term metabolic consequences of early antibiotic exposure. Improved means of reliably detecting neonates with bacterial infection would reduce the need for empirical antibiotic exposure initiated based on nonspecific symptoms and signs or risk factors. Finally, means to support healthy microbial contact in neonates and infants requiring antibiotic treatment are needed. The increasing prevalence of childhood obesity is one of the greatest challenges facing medical professionals caring for infants and children. According to a recent report from the United States, more than one in six children and youths between the ages of two and 19 years are obese and more than one in three are overweight . The detOverweight and obesity are highly hereditary because of both genetic predisposition and learned behavioral traits. However, hereditary factors fail to explain the massive increase in the prevalence of obesity in populations across the globe. The simple proximal explanation for obesity is excessive energy intake in comparison to energy expenditure. The obesity epidemic may accordingly be explained by changes related to lifestyle which have occurred during the past decades - our increasingly sedentary way of life combined with the all-to-common Western diet high in fat, carbohydrates and energy but low in fiber. Recent advances in scientific research have revealed intriguing mediating factors between the diet, host energy metabolism and the obese phenotype. Namely, the indigenous intestinal microbiota has been suggested to not only be influenced by diet but also to play a causal role in the development of obesity (reviewed in ).The association between the composition of the intestinal microbiota and obesity has been demonstrated by studies showing differences in gut microbiota composition between obese and lean humans , 6 and mvia microbial metabolites including short chain fatty acids [reviewed in 4], the production of which is influenced by dietary substrates. The low-grade inflammation characteristic of obesity and metabolic disease may also be induced and propagated by interaction with certain intestinal microbes [Bacteroides fragilis increase the risk of obesity development [via modulation of the gut microbiota [The direct causal role of intestinal microbes in the development of overweight and obesity has been demonstrated in experimental studies in which the obese phenotype may be transferred to germ-free animals merely by colonizing them with the gut microbiota from obese animals  or humanmicrobes –16. In pmicrobes , 16, wheelopment . It is telopment . Converscrobiota . The purAntimicrobial agents are among the most frequently used pharmaceuticals in infants and children. Cox and Blaser have recently estimated, that by two years of age, children in the United States have on average received nearly three courses of antibiotics and the number of courses increases to approximately ten by the age of ten years . It is lClostridium difficile, is a relatively common complication of treatment. Nonetheless, there are data suggesting that more prolonged gut microbiota alterations may result from repeated antibiotic exposure [Given the mechanism of action of antimicrobial agents, it is hardly surprising that antibiotic therapy inflicts a profound impact on intestinal microbial ecology , 24. In exposure . ConsideBifidobacterium species at four weeks of age as compared to non-exposed neonates [via breast milk are currently not known. In preterm infants, empirical antibiotic therapy based on a clinical suspicion of infection in the first week of life resulted in a higher relative abundance of Enterobacterium species and lower gut microbiota diversity at the age of three weeks [Enterobacterium species in particular is not known. Nonetheless, there are compelling data indicating that early life antibiotic exposure markedly increases the risk of overweight and obesity in later life.Antibiotic exposure in the neonatal period has a considerable impact on early gut microbiota development. In a study of nine term neonates subjected to treatment with ampicillin and gentamicin, this antibiotic exposure was associated with an increase in fecal Proteobacteria and a decrease in Actinobacteria and particularly neonates . The higneonates , who fouee weeks . The clip = 0.029). Interestingly, boys may be more affected by early antibiotic exposure than girls [A number of well-conducted epidemiological studies suggest that antibiotic exposure in infancy is associated with a higher body mass index (BMI) , 30 as wan girls , 32. Antan girls  but the an girls . These eper se and not antibiotic use increases the risk of psoriasis in the pediatric population [A potential confounding factor in the reports indicating an association between early antibiotic use and subsequent overweight or obesity are the infections because of which the antibiotic therapy was initiated. The large epidemiological studies published thus far have based their estimates of antibiotic exposure either on prescription records –33 or papulation . SimilarA substantial number of subjects are exposed to antimicrobial therapy without evidence of infection during the perinatal period . The widIt is well-established that other disturbances in perinatal microbial contact may increase the risk of obesity. Infants born by caesarean section (CS) delivery do not acquire the physiological inoculum of colonizing microbes from the birth canal and maternal gut that vaginally born infants receive. Substantial differences in gut microbiota between infants born by CS or through the vaginal route have been reported in early infancy . In the The strongest corroboration for the hypothesis of early antibiotic exposure causally increasing the risk of later-life obesity stems from experimental studies conducted using animal models. A series of experiments conducted in Dr. Blaser’s laboratory at the New York School of Medicine has demonstrated that exposure to low-dose antibiotics from weaning results in increased fat-mass accumulation in mice . This inA subsequent report from the same laboratory has further increased our understanding of these phenomena . MaternaThe association and possible causal relationship between early-life antibiotic exposure and the development of overweight and obesity raise concerns about current medical practices but may also provide opportunities to combat the obesity epidemic. It is clear that antibiotics are often used for the treatment of conditions, which are known to primarily be of viral origin or in instances where there is little or no evidence of efficacy. Prudent use of antibiotics is paramount to limit the propagation of antimicrobial resistance in pathogens. Fortunately, there are data indicating that antibiotic use in children has reduced during recent years . ResearcStreptococcus [According to current recommendations, newborn infants are to be treated with empirical antibiotic therapy based on non-specific symptoms and signs of infection or if the risk of infection has been estimated high based on factors such as maternal chorionamnionitis or colonization with group B tococcus . These pAntibiotic therapy is often a necessary and life-saving intervention in infants and children suffering from bacterial infection. Even with improved diagnostic methods and prudence in assessing the need for antibiotics, a considerable number of subjects need to be exposed to these drugs. To minimize the long-term risks involved with early antibiotic exposure, adjunct therapies aiming to alleviate its detrimental impact on the gut microbiota and consequently host metabolism should be developed. Oral bacteriotherapy with probiotic organisms might offer a means of modulating both the intestinal microbial community and host physiology. The efficacy of probiotics is well-established in the prevention of antibiotic-associated diarrhea . Future


The characteristic of HIV/AIDS Romanian epidemic is the high proportion of youth infected in 1988-1990, during their first year of life, when a nosocomial epidemiological accident took place. In Galati (South-East Romania) they were recorded 535 people with HIV/AIDS during 1990-2003 and 79.5% belonged to the pediatric nosocomial cohort. The mortality was 56.8% and half of the deaths were recorded during the first year after diagnosis. The aim of the study is to identify the changes in the HIV/AIDS epidemic in the last 10 years.This retrospective study was based on epidemiological reports of the newly diagnosed HIV/AIDS patients. The descriptive statistics and comparative Kaplan-Meier graphs by transmission way were analyzed using XL-Stat Software.During 2004-2013, 182 new patients have been diagnosed with HIV/AIDS in Galati. Most patients are young people with the same age as the pediatric cohort of HIV/ AIDS, but predominance of heterosexual transmission marks the change of the epidemic pattern. (table The life expectancy of HIV/AIDS patients has obviously improved in the last decade, but the yearly number of new cases is constant, with a high rate of late presenters. The strategy of future screening programs should consider the trends of HIV/AIDS epidemic in our region.


The objective of this study was to evaluate the appropriateness of antibiotic suspensions use for pediatrics among Palestinian mothers’ including their reconstitution, dose administration, duration, and storage conditions.This study was a questionnaire based cross sectional descriptive study. It was conducted at a governmental primary healthcare center and a pediatric private clinic in Nablus city between January and March 2013. A sample of 400 mothers were met and asked to answer a face to face questionnaire.The results showed that 347 (86.8 %) of mothers told that they read instructions, 311 (77.8 %) could understand manufacturers’ instructions, and 176 (44.0 %) of mothers asked pharmacists for advice when instructions were not well understood. In order to prepare antibiotic suspension, 302 (75.5 %) used boiled then cooled water, 192 (48.4 %) of mothers used a syringe to measure the needed amount of water, and 304 (76.6 %) added water in two steps, 392 (98.0 %) of mothers claimed that they shook the suspension bottle before use. Regarding dose administration, 313 (78.2 %) considered syringe as the most accurate tool for dose administration, most of mothers told that they gave drug dose with major meals when direction were to give three times daily, about use duration only 6 (1.5 %) mothers said that they used antibiotic suspension after 2 weeks, and 53 (6.5 %) gave left over antibiotic suspension to another child. One hundred seventy seven (44.2 %) mothers told they stored dry powder antibiotic in medicinal cabinet, while 226 (56.5 %) of them stored suspension in refrigerator.The results reflect a good level of correct practice. However, there is a room for improvement. The pharmacists are recommended to explain the correct directions, to supply a syringe with suitable calibration for dose administration, and to counsel parents about suitable storage condition, frequency of dosing and duration of use. Pediatric infectious diseases either viral or bacterial remain a very common community health problem with an average occurrence of 6–8 times a year . When inOral liquid medications usually come with a dose delivery device such as medication cups, droppers, calibrated spoons, and syringes . HousehoStorage conditions are also important, manufacturer instructions should be followed exactly, manufactures’ instructions recommend that some antibiotic suspensions need refrigerator, while for other antibiotic suspensions we need to avoid refrigerator.Antibiotics are misused because many patients do not take them according to their doctor or pharmacist instructions. They may stop taking their antibiotics too soon, before their illness is completely cured. Some patients save unused medicine and take it later for another illness, or pass it to other ill family members or friends and some patients go to the pharmacy and take antibiotics as an over the counter drugs .The aim of this study was to evaluate possible problems associated with antibiotic suspensions use among Palestinian mothers including their reconstitution, dose administration and storage conditions.This study was a questionnaire based cross sectional descriptive study. It was conducted at a governmental primary healthcare center and a pediatric private clinic in Nablus city between January and March 2013. A convenience sample of 400 randomly selected mothers was included. All mothers who were prescribed antibiotic suspensions for their children in the previous visit were asked to answer a face to face questionnaire until the required sample was met. The sample size was calculated based on Raosoft sample size calculator. Data collection tool was a questionnaire designed based on extensive literature review of similar studies –8. The qAmong 450 mothers approached, 400 women accepted to participate in the study giving a response rate of 88.9 %. Women age was mainly between 20 and 30 years 258 (64.5 %), with a mean age of 28.8 ± 6.2 years, range (17–52 years), 307 (76.8 %) were from Nablus city, 236 (59.0 %) of them had a child in the age group 1–3 years. Most of participants had high school or university degree 39.8 and 47.8 % respectively, 70.8 % of mothers had medical insurance, 68.5 % of participants reported medium monthly income, and 79.2 % were not working (Table During the study period the doctors were mainly visited due to throat infection (pharyngitis) 110 27.5 %), bronchitis 110 (27.5 %), and otitis media 108 (27 %)  claimed that they read the manufacturer instructions either on the box or in the package insert, 311 (77.8 %) of them told that they could understand the instructions. In case of not understanding the instructions, 176 (44.0 %) of participants asked for help from pharmacists, while 41 (10.2 %) asked their doctors  used boiled then cooled tap water, 52 (13.0 %) used mineral water, 31(7.8 %) used tap water directly, 7(1.8 %) used distilled water, and 5 (1.2 %) of drugs were prepared by pharmacists Table . FortunaRegarding the tool used to measure the volume of water for reconstitution, 192 mothers out of 397 who answered the question (48.4 %) used a syringe, while 179 (45.1 %) of participants used line on drug bottle, 25 (6.3 %) used enclosed medicinal cup, and 1 (0.3 %) used baby bottle  of mothers.Most of the mothers 392 (98.0 %) claimed to shake the drug bottle before use which means that 98.0 % of mothers followed correct practice.Among 400 mothers, 313 (78.2 %) of them considered syringe as the most accurate tool for drug administration, 57 (14.0 %), 20 (5.0 %), and 10 (2.5 %) of them considered household teaspoonful, medicinal spoonsful, and medicinal cup as the most accurate tool respectively, the correct practice according to related studies was practiced by 313 (78.2 %) of mothers.The mothers were asked about their practice if the direction for antibiotic suspension is to use three times daily; 224 (56.0 %) of mothers told that they would administer the dose with major meals as shown in Fig. Regarding the duration of administration; 394 (98.5 %) of mothers did not used suspension after 2 weeks of reconstitution, and 374 (93.5 %) of mothers did not use the leftover (remnant) for another child. Fortunately the correct practice according to manufacturer instructions was followed by 394 (98.5 %) and 374 (93.5 %) respectively.Mothers were asked about the storage of dry powder; 177 (44.2 %) of mothers told that they stored dry powder antibiotic in medicinal cabinet, 72 (18.0 %) in kitchen, 56 (14.0 %) in refrigerator, 48 (12.0 %) in dining room, 22 (5.5 %) above refrigerator, and 25 (6.2 %) of mothers used antibiotic directly, and the correct practice  was followed by 202 (50.5 %) of mothers.Storage of suspension after reconstitution; 226 (56.5 %) of mothers told that they stored antibiotic suspension in refrigerator, 71 (17.8 %) in medicinal cabinet, 54 13.5 %) in kitchen, 31 (7.8 %) in dining room, and 18 (4.5 %) above refrigerator  of 400 mothers read the instructions either on box or package insert, and 311 (77.8 %) told that they understood the instructions. This is a good level of awareness and understanding compared to others Palestinian studies, a previous study showed that 100 (45.0 %) of 222 consumers reported that they always read information in the leaflet of the drug package . It is rReconstitution of dry powder antibiotic depends on type of antibiotic and manufacturers’ instructions. Generally distilled or boiled then cooled tap water is used. First the bottle should be tabbed few times to loose the powder, then approximately half volume of water should be added, the bottle is shaken vigorously, the remaining of water should be added and shaken well. One exception is zinnat (cefuroxime) where the manufacturer recommends addition of water in one step. In this study 302 (75.5 %) of mothers used boiled then cooled tap water, 7 (1.8 %) of mothers used distilled water, this is a correct practice. While 52 13.0 %) used mineral water, and 31 (7.8 %) used tap water directly, this is a wrong practice. As mineral water is usually rich in minerals, this may cause decomposition of drug and complexion reactions .0 % used, while mRegarding the volume of water required to reconstitute the powders, the correct practice in this study was followed by 344 86.6 %) of mothers. In a previous Palestinian study, 3.5 % said the quantity of water that should be added to prepare suspension should not be a specific quantity, and 10.1 % could not decide or had no idea wither it should be specific or not [.6 % of mAppropriate dose administration includes shaking the bottle before use, and using accurate delivery tool, and dose interval. In this study most of the mothers 392 (98.0 %) claimed to shake the drug bottle before use. In a study from Nigeria, among 107 mothers 24.4 % did not shake the bottle before measuring the dose volume . In factAccording to this study 313 78.2 %) of mothers considered syringe as the most accurate device for dose administration, while 57 (14.3 %) considered household teaspoonful as the most accurate device for dose administration. The correct practice was followed by most of the mothers. A Ghanaian study showed that 95.0 % of 97 respondents used household spoons in dosing oral liquid medications 8.2 % of . CliniciRegarding to dosing interval a high percentage of mothers 224 (56.0 %) assumed that “use three times daily” means to give the drugs with major meals and this is a wrong practice. As we know three times daily for antibiotics means to administer the medication every 8 h. Regarding to this point, the role of pharmacists is very important and they should write the proper instructions and confirm on them during dispensing to avoid antibiotic failure or toxicity if the dosing interval are not followed properly.According to manufacturers’ directions antibiotic suspensions should be discarded after 10 days of reconstitution for antibiotics as amoxicillin, cefidinir, and cefuroxime, while amoxicillin–clavulanic acids, erythromycin, cephalexin should be discarded after 7 days, and after 5 days for azithromycin. This practice was followed by most mothers 394 (98.5 %). Regarding the use of left over suspensions, 26 (6.5 %) of mothers used left over antibiotic suspensions. Another study showed that 15.0 % of parents gave left over antibiotic suspension to their children which means that they keep antibiotics after reconstitution [Storage conditions of drugs are important as drugs are chemicals that may react with external environment such as temperature, humidity, and light. This leads to changes in drugs concentration properties and therapeutic effects. Many people stored medications in kitchen or bathroom cabinet, that speeds up medication breakdown process; as in both conditions drugs may be exposed to humidity and high temperature. Instead medicinal cabinet should be placed in a cool and dry place, away from direct sun light, and out of reach from children. In this study a good percentage of mothers 177 (44.2 %) stored dry powder antibiotic in medicinal cabinet, and 226 (56.5 %) of them stored antibiotic suspension in refrigerator. In both cases around half of the mothers followed the correct practice. These results are close to results from another Palestinian study which showed that 40.6 % of drugs were stored in pharmacy cabinet; however, the same study showed that 70.0 % of the reconstituted suspensions were stored outside the refrigerator . RegardiTo achieve higher level of awareness and correct practice regarding antibiotic suspensions use, cooperation between drugs companies, pharmacists, and doctors is needed. Drugs companies are recommended to write instructions in a simple way, and may draw the directions on bottle, in addition to providing suitable dose administration devices. The prescribers should inform mothers the correct instructions also and confirm that antibiotic suspensions should not be shared between children. It is the responsibility of pharmacists to explain instructions to mothers and confirm on them by writing, and to provide mothers with syringes, also it is important to concentrate on duration of use and storage conditions.The first limitation of this study is that the answers reported by the respondents cannot be validated and recall bias is possible, but this cannot be avoided in survey studies. Another limitation is the study was performed in one city only so it might not be representative to the practice in other places. However, these results can give a baseline data that can be useful in designing and implementing suitable educational programs and performing other related studies.The results reflect a good level of correct practice. However, there is a room for improvement. The pharmacists are recommended to explain the correct directions, to supply a syringe with suitable calibration for dose administration, and to counsel parents about suitable storage conditions, frequency of dosing and duration of use.


RNA viruses rapidly accumulate genetic variation, which can give rise to synthetic lethal (SL) and deleterious (SD) mutations. Synthetic lethal mutations  have been studied to develop cancer therapies. This principle can also be used against fast-evolving RNA-viruses. Indeed, targeting protein sites involved in SD + SL interactions with a drug would render any mutation of such sites, lethal.+ individuals: treated with drugs (T) or not (NT). Using a series of statistical approaches, we were able to propose bona fide SD + SL couples. When focusing on spatially close co-variant SD + SL couples at the surface of the protein, we found 5 SD + SL groups (2 in the protease and 3 in the reverse transcriptase), which could be good candidates to form pockets to accommodate potential drugs.Here, we set up a strategy to detect intragenic pairs of SL and SD at the surface of the protein to predict less escapable drug target sites. For this, we detected SD + SL, studying HIV protease (PR) and reverse transcriptase (RT) sequence alignments from two groups of VIHThus, designing drugs targeting these specific SD + SL groups would not allow the virus to mutate any residue involved in such groups without losing an essential function. Moreover, we also show that the selection pressure induced by the treatment leads to the appearance of new mutations, which change the mutational landscape of the protein. This drives the existence of differential SD + SL couples between the drug-treated and non-treated groups. Thus, new anti-viral drugs should be designed differently to target such groups.This article was reviewed by Neil Greenspan Csaba Pal and István Simon.The online version of this article (doi:10.1186/s13062-015-0044-y) contains supplementary material, which is available to authorized users. Classical SL genetic interactions involve non-lethal mutations (carried by two or more genes) whose combination leads to cell death. They have been extensively used to study gene-product interactions in the secretion pathway of yeast  and bactVarious studies have been performed to describe pair-wise and higher-order site correlations within RNA-virus proteins -17 emploin silico tool that predicts potential therapeutic targets. This tool has been tested on two HIV proteins, the protease (PR) and the reverse transcriptase (RT) and allowed us to describe five targets consisting of SL and invariant positions that should greatly minimize the emergence of drug resistance.In a preliminary work taking the HIV protease as a model, ,21,22, wet al.  was found not to be critical for function. For an antibody of known antigen specificity, Sharon mutated the Trp to Ala by site-directed mutagenesis without apparent effect on antibody reactivity for antigen.We agree with this comment. However, although we cannot say that every invariant site is essential for protein function, the contrary seems to be a special and not very widespread situation. All invariant amino acids or group of invariance (SL) cannot all be in this case. So, most of “invariant + SL” group are supposed underlie essential functions.3. Another assumption critical to the authors’ thesis is that drugs able to bind the sites identified as including SL/SD and invariant residues will effectively inhibit function for either PR or RT. I would not be surprised if some such drugs would exhibit disappointing levels of inhibitory activity, so that mutational escape would not be essential for the virus to continue to replicate.It could indeed be the case, as it has been in the past for many other inhibitors whose development does not, however, relied on the strategy described in this paper. We can not predict in advance the strength of an inhibitor. Moreover, our method describes the target and not the inhibitors themselves.et al. that evolutionary history may confound the identification of SL/SD amino acid pairs.4. Experimental testing will also help to address the concern raised by reviewers of the 2010 article by Brouillet It is thanks to the previous reviewer proposals that we set up a new test to select pairs of residues that interact for functional reasons and not for sharing a common ancestor (see answer to question 1). However, experimental testing is beyond the scope of this paper.et al. Nat Rev Genet. 2006 Feb;7(2):98–108].5. The authors appear to assume that synonymous mutations are selectively neutral. There are precedents for synonymous mutations that affect fitness through effects on RNA structure that influence the rate of translation or through other mechanisms [e.g., see Science. 2007 Jan 26;315(5811):525–8; reviewed in Chamary We agree with the comment of the referee. Synonymous mutations may affect RNA secondary structure, and even (indirectly) protein translation and conformation.How does this affect our results? Our method involves counting the non-synonymous and synonymous mutations per codon pairs. If the ratio is close to 1, we conclude that these codons are not subject to selection pressure and therefore the interdependence of residues comes from a shared common ancestor. False negatives could be obtained (ratio = 1). This requires the number of non-synonymous mutation pairs (numerator) selected by drug pressure to be similar to the pairs of synonymous mutations. Although this is possible in principle, we believe this phenomenon is less frequent than a direct impact of a mutation on the protein sequence. However, this deserves exploration in a further study.Reviewer 2: Csaba Pal, Biological Research Center, Hungary.The main objective of the paper is to identify intragenic pairs of residues that show synthetic lethal interactions in HIV proteins. The authors use this information to uncover drug target sites that could potentially mitigate the evolution of resistance. The manuscript is well written and the presentation of ideas goes straight to the point. The strategy followed by the authors is, to my knowledge, innovative and a valid approach to try to overcome drug resistance during HIV therapy.In fact, such approach, due to its target specificity and efficiency, would also be beneficial to the development of therapeutic approaches with less toxic side effects to the therapy of new-borns, infants and young children, which, together with multi-drug resistance, is an important problem to be solved in HIV therapy. The idea of creating a software tool for the identification of inescapable drug targets is very important, and could help medicinal chemists to focus their research on compounds that bind to the predicted target sites.The authors should discuss the benefits and future perspectives of the work more deeply in the paper. For example, the possibility to apply such methodology to other target proteins should also briefly be discussed in the manuscript. The authors should also discuss in vitro/in vivo validation of the reported results, including possible limitations of such studies.This method, which is based on SL approach and not through the development of competitive inhibitors, could enable the discovery of less toxic molecules, which are necessary to treat more vulnerable patients.We suggest that it would be possible to do similar studies on other RNA virus proteins.We also explain the biological tests that will be required to validate the method and the limitations of such techniques.We thank the referee for these suggestions and will accordingly change the manuscript:These changes will be highlighted in yellow in the text.Reviewer 3: István Simon, Institute of Enzymology, Hungary.This paper is a follow-up of a few papers by Anne Vanet and coworkers on synthetic lethals. I recommend its publication after some revisions. First, the authors should clarify what the novel findings of this paper are.(See the reply to reviewer 1 comment 1).Also the druggable nature of a target should be checked by in silico docking, using large drug datasets and fast docking programs.in silico checking cannot be done at the occasion of a minor addition of the paper. Indeed, it should be the focus of a full scientific project.We thought to check the druggable nature of the target with in-silico docking using large datasets. However, as far as we know, all currently available docking programs need to initialize crucial parameters before being launched. Among them, there are the initial location and orientation of each chemical in the cartesian coordinates system of the target. The optimal selection of these parameters depends both on the target and on the ligand. It means that writing the script to launch the docking is a complex task, which may not be immediately successfull. Moreover, even when working on known target-ligand complexes, it is known that, most of the time, the correct pose is not retrieved as the first one ranked by the docking software, and this correct pose is more likely to be found among the ten or the twenty best poses, if ever found. Their manual analysis  is difficult even for a small number of chemicals, so the automatic analysis of the results for large datasets is a complex task, too. Thus we feel that the requested There are some minor issues. The reference of the sequence data and not only the URL should be given.We will provide the reference of the sequence dataset.Intervirology 2012;55(2):98–101. Epub 2012 Jan 24.Tang MW, Liu TF, Shafer RW. (2012). The HIVdb system for HIV-1 genotypic resistance interpretation. Also the reference for Pymol should be included.The version used in this work is MacPyMOL0.99 To our knowledge, this software has never been published.It is reasonable that some drug, which fits to the PR-NT and RT-NT cases can not be used for PR-T and RT-T cases, but it is not clear why they are not usable the other way around.We agree with the referee, the opposite case is more complicated to understand. In fact, we must keep in mind that the pairs of SL and CM are embedded within a complex network of pairs that evolves when an individual node of the network changes. Thus, in the treated patient sequence sets, several mutations occur in response to the selection pressure and the drugs developed for untreated patients may no longer work for most patients. These new mutations could cause a modification of the mutational landscape and reveal new pairs of SL, of course only in the treated patient sets. New drugs based on these SL should therefore be effective on treated patients and not on untreated patients.et al. Bioinformatics 28 (15) 2074–2075; 2012 to learn how to do it without prior knowledge of the binding sites.I understand that the authors are not willing to make the docking calculations in the present paper, so the paper can be published without it. However if they are willing to do it in a later paper, I suggest to consult the paper: Volkamer A. Also, I would like to call their attention to the notice of the current distributor of PyMOL:http://www.pymol.org/citingLike many software programs PyMOL was not published. It does not have a scientific algorithm one can publish. Still it is necessary to cite it. You can find instructions on how to do that here: I suggest to consider it even for this paper.


Intensive care units (ICU) are epicenters for the emergence of antibiotic-resistant Gram-negative bacteria (ARGNB) because of high rates of antibiotic usage, rapid patient turnover, immunological susceptibility of acutely ill patients, and frequent contact between healthcare workers and patients, facilitating cross-transmission.Antibiotic stewardship programs are considered important to reduce antibiotic resistance, but the effectiveness of strategies such as, for instance, antibiotic rotation, have not been determined rigorously. Interpretation of available studies on antibiotic rotation is hampered by heterogeneity in implemented strategies and suboptimal study designs. In this cluster-randomized, crossover trial the effects of two antibiotic rotation strategies, antibiotic mixing and cycling, on the prevalence of ARGNB in ICUs are determined. Antibiotic mixing aims to create maximum antibiotic heterogeneity, and cycling aims to create maximum antibiotic homogeneity during consecutive periods.This is an open cluster-randomized crossover study of mixing and cycling of antibiotics in eight ICUs in five European countries. During cycling (9 months) third- or fourth-generation cephalosporins, piperacillin-tazobactam and carbapenems will be rotated during consecutive 6-week periods as the primary empiric treatment in patients suspected of infection caused by Gram-negative bacteria. During mixing (9 months), the same antibiotics will be rotated for each consecutive antibiotic course. Both intervention periods will be preceded by a baseline period of 4 months. ICUs will be randomized to consecutively implement either the mixing and then cycling strategy, or vice versa. The primary outcome is the ICU prevalence of ARGNB, determined through monthly point-prevalence screening of oropharynx and perineum. Secondary outcomes are rates of acquisition of ARGNB, bacteremia and appropriateness of therapy, length of stay in the ICU and ICU mortality. Results will be adjusted for intracluster correlation, and patient- and ICU-level variables of case-mix and infection-prevention measures using advanced regression modeling.This trial will determine the effects of antibiotic mixing and cycling on the unit-wide prevalence of ARGNB in ICUs.NCT01293071 December 2010.ClinicalTrials.gov  Infections caused by Gram-negative bacteria frequently complicate treatment of critically ill patients in the intensive care unit (ICU). Such ICU-acquired infections are associated with higher morbidity and mortality. The sevAs a response, antibiotic stewardship programs aim to optimize the rational and prudent use of antibiotics. These programs attempt to reduce selective pressure by reducing overall antibiotic consumption, but also to optimize the choice of the antibiotic, dosing and administration route. As such, antibiotic rotation has been proposed to reduce antibiotic resistance through systematically rotating antibiotics or antibiotic classes for empirical treatment.There are two types of rotation schemes: mixing and cycling. In mixing, the treatment is changed with every new antibiotic course, and in cycling, empiric antibiotics change per time block (weeks or months). These interventions have been evaluated in ICUs, but also in neonatal-, pediatric-, oncology- and cardiothoracic-surgery departments-23. The This multicenter cluster randomized crossover trial was designed to determine the effects of mixing and cycling of antibiotics in eight European ICUs, incorporating the most relevant confounders and adjusting for clustering of results in the analysis. For uniformity of reporting, we have used the CONSORT 2010 statement: extension to cluster randomized trials.The primary objective of this trial is to compare the effects of a strategy of antibiotic mixing  to a strategy of antibiotic cycling  on the mean unit-wide prevalence of antibiotic-resistant Gram-negative bacteria (ARGNB). The primary hypothesis is superiority of one intervention arm over the other.The trial has a cluster-randomized, crossover design. All ICUs start with a 4-month standard care period with no interventions and are then randomized to one of two interventions of 9 months. After a wash-out period of 1 month, the ICUs cross over and perform the alternate rotation strategy for a second 9-month period , Germany (n = 2), Portugal and Slovenia (n = 2)). For all ICUs, IRB approval that required a waiver for individual patient written informed consent was obtained. The SATURN ICU trial was registered in the ClinicalTrials.gov (NCT01293071).1. At least eight beds with capacity of mechanical ventilation2. Presence of at least one research nurse  dedicated to the trial3. Facilities for storage of screening swabs at -70 degrees Celsius4. Approval of study protocol by the local Institutional Review Board (IRB)5. Ability to obtain an informed consent waiver for individual patients6. Ability to obtain written consent by physician and nursing staff representative to participate in the trial7. Availability of a digital data patient management system for data extraction8. No planned implementation of other interventions that may affect resistance prevalence during the study period9. Not being an Intensive Care Burn Unit, Cardiothoracic Surgery Unit, Pediatric Intensive Care Unit, or Neonatal Intensive Care UnitDuring the intervention periods, the ICU-wide preferred empirical treatment for ICU-acquired infections with Gram-negative bacteria is rotated according to two protocols; mixing and cycling. The three rotated antibiotics will be 1) third- or fourth- generation cephalosporins, 2) piperacillin-tazobactam and 3) carbapenems.During mixing, the preferred choice for empiric antimicrobial treatment changes with every newly prescribed empiric antibiotic course. During cycling, empiric treatment changes every 6 weeks. The order of interventions and the order of rotated antibiotics are randomized at the beginning of the trial by a person not involved in the design or execution of the study.Treating physicians can deviate from study protocol at any point and for any reason because of patient safety or as part of de-escalation of antibiotic treatment. Combination therapy with preferred antibiotics (such as addition of an aminoglycoside or coverage of Gram-positive bacteria) is allowed. ICU-specific procedures related to standard hygienic measures, monitoring practices, outbreak management and any other infection prevention measures will not be dictated by study protocol.Overall antibiotic use will be derived from ward-level consumption based on either individual prescription data or weekly unit level administrative antibiotic data.As a proxy for protocol adherence, weekly point-prevalence measurements of antibiotic consumption will be taken. Antibiotic courses are then counted, regardless of dose or route of administration. The counts will then be translated to fractions of study-adherent antibiotics of all antibiotics. With optimal adherence, the three preferred antibiotics should be in equal proportions during mixing, whereas the preferred antibiotic should be dominant during cycling. Weekly graphs of ICU-specific results are then returned to each of the ICUs to provide compliance feedback.Pseudomonas aeruginosa and Acinetobacter species, and Carbapenem resistance in P. aeruginosa and Acinetobacter species . Resistance is defined as extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, Piperacillin-Tazobactam resistance in Enterobacteriaceae, s Table .Secondary endpoints are: 1) individual patient acquisition rates of ARGNB per admission days at risk; defined as the first positive culture for ARGNB >48 hrs after admission. 2) ICU-acquired bacteremia rates with ARGNB per admission days at risk; 3) percentage of appropriate empirical treatment of ICU-acquired bacteremia; 4) mean length of stay in ICU; and 5) mean in-ICU-mortality.For these secondary outcomes, clinical culture results will be used to determine ARGNB colonization status. Appropriateness of empirical treatment will be based on blood culture isolate antibiograms and antibiotic treatment prescribed. Length of ICU stay and in-ICU mortality will be derived from computerized patient records.Sample size calculations for individually randomized patients assume that patient outcome is independent of other patients’ outcomes, an assumption that is frequently violated when investigating the dynamics of infectious disease. Ignoring this interpatient dependency may lead to overestimation of treatment effect and underestimation of the necessary sample size.The calculated sample size without clustering is 392 cultured patients per intervention arm, assuming a binomial distribution and based on 80% power to detect an absolute change of 10% in resistance prevalence with a 95% confidence level using a two-sided test. The sample size is based on a worst-case scenario with regard to precision; the widest distribution and thus largest needed sample size is around 50% for a binomial distribution. Therefore, the prevalence decrease was set from 55% to 45%. As an illustration, to detect a reduction from 30% to 20%, 291 patients per intervention period are needed. To include clustering effects we used an Intra-class Correlation Coefficient (ICC) of 0.01, based on a previous cluster-randomized ICU study. For estWith an expected 2,160 sampled patients for both intervention arms , our study should therefore be adequately powered.Dedicated staff will collect data from  patient charts and microbiology reports. Screening swabs will be collected by qualified personnel, either by the ICU-staff or study-nurse. Data of potential confounders is collected from computerized individual patient records . Ward-specific confounder data  are obtained from monthly point-prevalence measurements.Weekly point-prevalence measurements of antibiotic consumption will be collected either directly on the ward or from the patient data management system.Two types of outcome analysis will be performed: analysis of the ICU-level of resistance prevalence (primary outcome) and analyses on individual patient level, including the secondary outcome measures Table .Descriptive analysis will be performed of baseline characteristics and covariates. Where appropriate, bivariate tests will be performed. Bivariate statistical testing and advanced regression analysis will be used to model the effect of the interventions on resistance prevalence and acquisition. This will include adjusting, if necessary, for clustering of outcome data and confounding within ICUs by patient demographics, antibiotic consumption, illness severity, infection prevention measures and time trends. Results from point-prevalence measurement will be analyzed as continuous or count measurements per ICU for each intervention period, taking auto-correlation within ICUs into account in regression analysis.The primary outcome measurements will be analyzed using McNemar’s test for dependent pairs with all outcomes pooled for mixing or cycling. Bivariate tests, however, do not take into account differences in effects between hospitals, trends over time or possible confounding. Therefore, a stepwise mixed effects model will be constructed using the prevalence of resistance colonization as outcome, and the interventions as a factor. To assess differences in treatment effects between ICUs and time trends over study periods, random effects for the eight ICUs and the nine longitudinal measurements per ICU per study period will be added stepwise to the model. In the case of an imbalanced case-mix between interventions, confounders can be added to the model to adjust for these differences. This will facilitate adjustment for intracluster correlation and for imbalances in the study arm case mix (patient and ICU characteristics) not caused by the interventions themselves.For the secondary outcomes, analyses are stated in Table Because of the crossover design, period effects and carry-over effects will be assessed.The association between antibiotic use and selective pressure for ARGNB, together with the incomplete evidence base for antibiotic rotation strategies in ICU settings, warrants more research on this subject. The lack of a consistently applied methodology to study antibiotic rotation strategies, and the suboptimal designs applied in some studies, seriously hamper interpretation of available results at present. We haveWe decided to target currently important pathogens, ARGNB, and the antibiotics mostly used for those infections. Consequently, the used antibiotic classes will be all beta-lactam antibiotics . It could be hypothesized that rotation of antibiotics with different mechanisms of action would increase the effects of mixing and/or cycling. However, given the current increase in incidence of ESBL-producing bacteria and resistance to fluoroquinolones, choices of empiric antibiotics have become limited in the different regions of Europe. A too-specific choice of antibiotics would prevent inclusion of representative European ICUs, and would, therefore, reduce generalizability of findings. Indeed, the antibiotic strategies evaluated in this study may not be relevant for settings with much lower levels of ESBL-producing bacteria or in settings with endemicity of carbapenem-resistant Enterobacteriaceae.The optimal timing of cycling interventions is as of yet unknown, and periods of weeks to months have been used in previous studies,20-22,27We decided to use cycling periods of 6 weeks, to evaluate reintroduction of the intervention antibiotics. Six-week periods allow two complete cycles of three antibiotics in 9 months. The aim of this reintroduction is to investigate possibly faster re-emergence of antibiotic resistance during the second cycle of antibiotics.The monthly ICU-level, point-prevalence measurements will provide unbiased data for ARGNB prevalence. Furthermore, clinical culture results provide additional specific, though less sensitive, colonization incidence data.The open cluster randomized design with a baseline period and crossover of interventions has methodological advantages but also limitations over the quasi-experimental before-after design or patient randomized studies.First, the cluster design, with interventions applied unit-wide, prevents allocation bias within ICUs and reduces contamination of intervention effects. With individual patient randomization, different interventions will be executed in the same ICU at the same time. The transmissibility of infectious disease implies that colonization rates for patients in the same ward will become dependent, which may well decrease outcome differences between interventions. A cluster-randomized study design physically separates the two intervention populations and thereby prevents transmission-based dependency in resistance outcomes. Possible associations between resistance acquisition within an ICU are now associated with only one intervention. The disadvantage is the introduction of clustering, or correlation of results within ICUs, which needs to be accounted for in sample size calculations and analysis.Due to the consecutive inclusions in cluster randomized studies, these are more susceptible to chance fluctuations potentially causing selection bias, which needs to be assessed and adjusted for, if present.Second, the open design may lead to allocation bias, where certain patient groups will not be eligible for the treatment with the preferred study antibiotic. Hospital specific distributions of parameters such as treatment indication and/or comorbidities will therefore influence adherence and possibly the effect of interventions, depending on the intervention phase and preferred antibiotic. The crossover design, however, will prevent differential distribution/bias between intervention groups. Nevertheless, the different interventions could have implications for adherence still causing bias between interventions with regard to intervention effect. Assessing differences in intervention adherence is therefore part of the analysis.Third, both baseline antibiotic use -and resistance prevalence are known to change over time, even without interventions such as antibiotic rotation. Baseline study periods allow quantification of the effects of both interventions on overall antibiotic use and resistance prevalence, and extend the possibilities for time-trend analysis, given the absence of an adequate parallel control group in time. It was decided not to include such a parallel control group because of the large differences of patient populations between ICUs, even within the same country.Fourth, the crossover of interventions provides adjustment for differences between units that may also affect the prevalence of ARGNB.Finally, with regard to outcome data collection, centralized processing of samples will prevent hospital specific differences in detection of resistance. Admission and discharge swabs are not obtained, making this study less suited to determine acquisition of cross-transmission rates of ARGNB.To our knowledge, this will be the first multicenter study with eight ICUs on these interventions aiming to include approximately 10,000 patients in five different countries. The powThe intraclass correlation coefficient (ICC) was used based on mortality data from a multicenter study in 13 Dutch ICUs. The ICC could therefore be different for antibiotic resistance data, which would change the power of our sample size. Nonetheless, with our current estimated sample size, the ICC could increase 60% to 0.016. In addition, this calculation does not account for the crossover design, ignoring the reduction in interclass correlation resulting from this design.Also, the sample-size calculation assumed a similar ICC for all ICUs, did not take clustering of results obtained on the same sampling day into account, and the ICC did not include a reported standard error, as recommended in the CONSORT statement.Outlining the analysis plan, descriptive analysis and exploration of covariates will precede final regression analysis. In general terms, the prevalence during both interventions and secondary outcomes will be compared, assessing whether any - and which of the two interventions - is superior over the other. The baseline periods will be used to compare standard care to the intervention periods. The predefined analysis will include bivariate testing and stepwise methods adjusting for possible influences of the study design and time effects. This includes clustering of outcome data, time trends and confounding using Generalized Linear Mixed Models. Secondary outcomes will be analyzed using bivariate testing, and Cox-proportional hazard models also accounting for random effects.Better understanding of the associations between antibiotic pressure and resistance emergence is needed and important. This trial will provide further insight in the use of mixing and cycling of antibiotics and guide future practice guidelines and clinical and mathematical modeling studies on the effects of antibiotic policies.Recruiting.Acinetobacter, Pseudomonas and Stenotrophomonas species will be sent to the Barcelona Centre for International Health Research (CRESIB), Barcelona, Spain for pheno- and genotyping.Screening swabs from monthly point-prevalence measurements will be taken from oropharynx and perineum of all patients in the ICU at that time point. Swabs will be put directly in medium and frozen at -70° Celsius. The swabs will then be shipped in batches under frozen conditions to the UMCU laboratory and centrally typed in the UMCU laboratory by dedicated laboratory analysts. Swabs will be cultured on five different plates Table . Plates ARGNB: antibiotic-resistant Gram-negative bacteria; CONSORT: Consolidated Standards of Reporting Trials; ESBL: extended-spectrum beta-lactamase; ICC: intra-class correlation coefficient; ICU: intensive care unit; IRB: institutional review board.The authors declare that they have no competing interests.PJD prepared the study protocol and this manuscript. MJMB was co-applicant for a European Union Framework Program 7 budget for this trial as part of the SATURN consortium, and also conceptualized the protocol and supervised preparation of the study protocol and manuscript. All authors read and approved the final manuscript.


Achondroplasia, Duchenne muscular dystrophy, and osteogenesis imperfecta are among the most frequent rare genetic disorders affecting the musculoskeletal system in children. Rare genetic disorders are severely disabling and can have substantial impacts on families, children, and on healthcare systems. This literature review aims to classify, summarize and compare these non-medical impacts of achondroplasia, Duchenne muscular dystrophy and osteogenesis imperfecta. A rare disease is defined as one that affects less than one in 2000 individuals in Europe and one in 1250 in the United States . DespiteThis paper reports a scoping review that describes the literature on non-medical impacts for patients and families. Due to the exploratory nature of the review and to reflect the research areas of our team, we delineated the focus of this review to three common RGDs in the pediatric orthopedic context: achondroplasia, Duchenne muscular dystrophy (DMD) and osteogenesis imperfecta (OI). These three RGDs are single-gene musculoskeletal diseases characterized by physical disability and little or no impairment of mental ability. Table The three specific objectives of this review research were: i) to categorize the types of non-medical impacts of achondroplasia, DMD and OI; ii) to summarize these impacts; and iii) to discuss findings on these impacts across the three diseases.While systematic reviews synthesize the complete nature of a particular field, outlining what approaches are effective and where further research is required, scoping reviews are exploratory projects that map the literature available on a topic, identifying the key concepts, theories, sources of evidence, and gaps in the research . We therFrom March to June, 2013, we conducted a search of the literature in three electronic databases: Web of Science, CINAHL, and MEDLINE. The following two string combinations of keywords were used:  AND . We complemented our search for publications with a systematic screening of the table of contents of specific journals dedicated to rare diseases  and a screening of references of relevant publications. Opinions, commentaries, letters, editorials, and publications without an abstract were immediately initially excluded. The initial search yielded the identification of 845 publications. After removing 8 duplicates, two authors independently selected publications addressing the non-medical impacts of Achondroplasia, DMD and OI, based on the titles and the abstracts. Publications that addressed a rare genetic disease other than the three retained by the research group were excluded from this review, as were publications focussed on the identification of genes. In the case of disagreement among authors, the full-text publications were reviewed by a third senior author. A total of 65 full-text publications remained at this stage of the review.For inclusion in our study, publications had to be i) published between 1980 and 2013; ii) in English or French; iii) about non-medical effects and responses to OI, achondroplasia and DMD; and iv) have a well-defined methodology. Because a scoping review is not intended to assess the quality of studies, emphasis was not placed on methodological rigor of the retained studies. At this second stage, 8 publications were excluded as they examined medical impacts of a treatment or intervention, and 57 publications were retained. Figure The 57 retained publications were coded using qualitative structure coding  with NviTo ensure consistent mapping and analysis of data, we developed a glossary of codes used to classify the impacts as follows: i) ‘scope’ refers to whether the article addresses one or several pediatric rare musculoskeletal disorders; ii) ‘direction’ was used to capture whether the article studied the ‘Effect of Disease’ (Direction 1) or ‘Response to the Disease’ (Direction 2); iii) ‘target’ refers to three distinct groups of focus: ‘individuals’ such as patients, adults, carers, or siblings; ‘groups’ such as physicians, caregivers, or school teachers; and ‘societies’ such as communities or society as a whole; iv) ‘timeline’ and ‘trend’ refer to the temporal analysis of impacts; and v) ‘type’ refers to positive or negative consequences; finally vi) ‘domain’ refers to the aspect of QOL being studied  or the research tradition or academic discipline used to study the impacts. Once the 57 publications were categorized, we performed a narrative review of the findings on the impacts separately for each disease, and then critically compared the findings across the three diseases. An overview of the distribution of the publications using the components of our working framework is presented, as well as a summary of the major findings of the publications achondroplasia, DMD and OI. Finally, we discuss how research compares across the three diseases.A total of 57 publications on non-medical impacts are included in this review study: 3 about achondroplasia -15, 39 aThe majority of the publications (50) were original research focused on a single disease, while 6 publications concerned two diseases (DMD and other diseases), and 1 publication was a literature review.The target group assessed was spread between the experiences of the individual with the disease (26), the impact of the disease on families (18), the experience of caregivers only (8), or were focused on the impacts of other groups or tool development and reviews of the literature .The direction of the impacts was explicitly stated in 44 of the 57: 31 addressed the effects and impacts of living with achondroplasia, DMD or OI, 13 addressed responses to living with the disease on individuals, groups or society . In these 44 papers, the occurrence of a RGD was conceptualised as a source of stress to which people react. The direction was either a combination of directions, or not explicitly stated, in the remaining 13 papers. Table Papers were also spread over several domains (as defined above), with 15 examining the quality of life (QOL) through a normative and validated measure of function, psychosocial well-being, and perception of health . Psychosocial impacts on their own were addressed in 18 publications , and functional status on its own was addressed in 10 publications . The remaining 14 publications assessed the domains of illness experience, utilisation of healthcare services, palliative care, medical staff reactions to the disease, or newborn screening. Tables Evidence from the publications on achondroplasia suggests that affected individuals have an impaired QOL when compared to first-degree relatives . The lowThe overall QOL of parents and their children who are affected with DMD was reported to be lower than in the general population, particularly due to impairments in physical functioning ,42. BoysThe 14 studies that solely examined the psychological impacts of DMD reported higher levels of anxiety, depression, and guilt in parents, particularly in mothers ,26,46. HIn addition to the psychological impacts, parents, particularly mothers, reported caring for their children with DMD to be burdensome , costly in terms of time, and contributing to increased social isolation . AltogetThere was little difference in the overall QOL of those diagnosed with OI as compared to the general population. Functional limitations were more important in patients with severe forms of OI, and higher life satisfaction, resilience, low depression and higher social achievements were common ,64,68. PBesides these psychosocial impacts of achondroplasia, DMD and OI on families, the impacts on groups and the society were broached in few studies. Two publications examined the attitudes of medical staff about a condition or related treatments options ,40. ThesThe impacts of achondroplasia, DMD and OI on families reported in this review are derived from in-depth qualitative analysis of subjective illness experiences or objective normative measures of QOL. These methodological specificities resulted in variations in the impacts between and within similar categories of publications that may limit the comparison across the three diseases. However, caring for children particularly with DMD and OI was burdensome because of daily practical problems , increased time costs and social isolation, as reported in studies on other RGDs -72. ConsAs for the QOL in patients with these three diseases, findings seem contradictory with some publications reporting higher QOL and other reporting lower QOL when compared to healthier unaffected controls ,22. The Some practical implications emerged from this review that can benefit families and caregivers. The correlation between improved parental coping strategies and early diagnosis, better emotional functioning of the child and siblings suggests that there have been efforts to obtaining an early diagnosis and promoting family hardiness for people living with these diseases ,75. To tFrom the limited number of publications on the impacts of DMD, achondroplasia and OI, we have learned that research about non-medical impacts of achondroplasia, DMD and OI on healthcare systems and society seems very limited and could be further addressed. However, the selection of the keywords used in the searching strategy in this review might have resulted in the exclusion of some publications. For example, challenges in providing adequate updating training to clinicians and staff on these rare diseases as acknowledged in other RGDs could be examined. In addition, research on healthcare-related impacts might compare the use of healthcare resources according to the severity of the disease. Efficient care delivery models organised around primary care whereby family physicians can help to achieve a smooth transition to adult care institutions must also be investigated ,77,78. IWe acknowledge that methodological limitations could have compromised the inclusion of publications reviewed here. Thus, further longitudinal studies combining normative approaches with qualitative in-depth investigations and comparing several diseases could enhance the understanding of patients’ effective coping strategies. Simultaneous use of parent-report and patient-report questionnaires could provide increased insights . FurtherIn general, DMD and OI negatively impacted carers. Some events seem particularly critical such as occurrence of fractures or loss of ambulation in patients. As for patients, functional limitations seemed to follow the severity of the disease, but psychological distress did not, which calls for a better understanding of effective coping strategies. This conclusion is supported by the reported higher life satisfaction in very severely affected patients at different stages of the progressing course of DMD, and the reported resilience characteristic of adolescents with OI. In face of the great difficulties presented to families, life span and family-centered psychological support must be developed in a timely fashion to aid patients, carers, and siblings.


The prognostic value of the copy number (GCN) and protein expression of the mesenchymal-epithelial transition (MET) gene for survival of patients with non-small cell lung cancer (NSCLC) remains controversial. This study aims to comprehensively and quantitatively asses the suitability of MET GCN and protein expression to predict patients' survival.PubMed, Embase, Web of Science and Google Scholar were searched for articles comparing overall survival in patients with high MET GCN or protein expression with those with low level. Pooled hazard ratio (HR) and 95% confidence intervals (CIs) were calculated using the random and the fixed-effects models. Subgroup and sensitivity analyses were also performed.p<0.001; protein expression: HR = 1.52, 95% CI 1.08–2.15, p = 0.017). In Asian populations , but not in the non-Asian subset. For adenocarcinoma, high MET GCN or protein expression indicated decreased OS . Results were similar for multivariate analysis . The results of the sensitivity analysis were not materially altered and did not draw different conclusions.Eighteen eligible studies enrolling 5,516 patients were identified. Pooled analyses revealed that high MET GCN or protein expression was associated with poor overall survival (OS) (GCN: HR = 1.90, 95% CI 1.35–2.68, Increased MET GCN or protein expression was significantly associated with poorer survival in patients with surgically resected NSCLC; this information could potentially further stratify patients in clinical treatment. Lung cancer continues to be the most common and deadly malignant cancers worldwide The MET gene, located at 7q21-q31, is a potential prognostic genetic marker, which encodes a receptor tyrosine kinase for the HGF/scatter factor (SF) With the aim to gain a better insight into the prognostic value of the copy number or protein expression of the MET gene for survival of patients with non-small cell lung cancer, we conducted the first comprehensive meta-analysis of published literature on this topic.PubMed, Embase, Web of Science and Google Scholar were searched for articles concerning the MET GCN, MET protein expression, disease status and survival in patients with NSCLC. The last search update was December 12, 2013. The search strategy included the following medical subject heading terms and keywords variably combined: “Proto-Oncogene Proteins c-met” [Mesh], “Carcinoma, Non-Small-Cell Lung” [Mesh], “MET”, “c-met”, “met Proto-Oncogene Proteins” “Hepatocyte Growth Factor Receptor”, “Scatter Factor Receptor”, “HGF Receptor”, “met gene copy number” “lung cancer”, “NSCLC”, “prognosis”, “prognostic” and “survival”. We did not apply any language restrictions.in situ hybridization (FISH), reverse transcription-polymerase chain reaction (RT-PCR), and immunohistochemistry (IHC); and (III) Findings providing sufficient information for the estimation of hazard ratios and 95% confidence intervals. Only studies published in peer-reviewed journals were included, data from letters and meetings abstracts were not eligible. Two researchers (B.P.G and H.C) independently screened and determined the relevant studies. Any discrepancies were settled through discussion until a consensus was reached.Studies meeting the following inclusion criteria were considered for this meta-analysis: (I) Clinical trials and prospective or retrospective cohort studies investigating the correlation of the MET GCN and protein expression status with the OS of NSCLC patients; (II) Measurement methods, including fluorescent Two reviewers independently (B.P.G and H.C) extracted the relevant data from each study and subsequently assessed the data to estimate reliability. The following information was obtained from the MET GCN studies: the first author, year of publication, country of origin, inclusion period, number of patients , age at time of diagnosis , tumor stage, method of MET GCN detection, cutoff value of high MET GCN, histology, number of patients of high MET GCN, treatment, time of follow-up , and OS data. The information obtained from each MET protein expression study included the first author, year of publication, country of origin, inclusion period, number of patients , age at time of diagnosis , tumor stage, method of MET protein expression detection, specimen, cutoff, antibodies, histology, number of patients of high MET protein expression, treatment, time of follow-up , and OS data.Two authors (B.P.G and X.H.T) independently assessed the quality of the selected studies using the Newcastle-Ottawa Scale for cohort studies (NOS) The primary results were stratified according to MET GCN (high vs. low) and protein expression (high vs. low). The HRs and 95% CIs were combined to obtain the effective value. When the HR was not reported in an article, this parameter was calculated using the methods of Parmar et al 2I and Q statistics was performed. The heterogeneity of individual HRs was calculated using Χ2 tests according to the method of Peto p value less than 0.10. 2I was used to quantify inconsistencies, where a value of 0% indicates no observed heterogeneity, a value less than 25% denotes low heterogeneity, a value from 25.1–50% indicates moderate heterogeneity, and a value greater than 50% indicates substantial heterogeneity A heterogeneity test based on p-value of less than 0.05 for summary HR and publication biases. All calculations were performed using STATA version 11.0 .Sensitivity analyses were performed to assess the stability of the results. Egger's test A total of 939 records were identified by the primary computerized literature search. After screening the titles and abstracts, thirty-one articles were further reviewed in detail. As indicated in the search flow diagram , 18 studFor MET GCN, most studies were retrospective cohorts and only one study was a prospective cohort. A total of 11 studies For MET protein expression, all eligible studies were retrospective cohorts. A total of 2,650 patients were included in 13 studies The study quality was assessed using the Newcastle–Ottawa quality assessment scale, generating scores ranging from 4 to 9 (with a mean of 5.85), with a higher value indicating better methodology. The results of quality assessment are shown in supplementary p<0.001) in eleven studies p = 0.005) in nine studies 2I = 58.0%, p = 0.008) and multivariate  analyses , and the pooled results remained practically unchanged .For OS, the estimated pooled HR for increased MET GCN, using univariate and multivariate analyses, was 1.90 , but these results were not observed for non-Asian populations  , the pooled HR for adenocarcinoma was 1.49 (95% CI: 1.05–2.10) and the combined HR for squamous cell carcinoma was 1.64 (95% CI: 0.54–4.60)  Table 3Table 3. = 0.630)  (Table 3= 0.630) .p = 0.0017), indicating that MET overexpression had worse survival impact in patients with NSCLC  was observed, we applied the random-effects model. One study 2I = 15.2%, p = 0.314), and the pooled results remained practically unchanged .The combined HR for the nine studies th NSCLC . Becausep<0.00)  was observed among these studies, a random-effects model was applied. The observed heterogeneity might reflect the difference in the populations studied and experimental methods used. Onisuka et al 2I = 25.9%, p = 0.24), and the pooled results remained practically unchanged .Eight studies p<0.00) . Becausep<0.001), but these results were not observed in non-Asian populations  , and the pooled HR for adenocarcinoma was 1.69 (95% CI: 1.31–2.19)  Table 3Table 3. = 0.623) .p = 0.352 for univariate analysis and p = 0.063 for multivariate analysis). For the MET protein expression, there was no evidence for significant publication bias .The sensitivity analysis indicated that omitting any single study did not influence the pooled HRs. For MET GCN, A more formal evaluation using Egger's test showed no evidence of significant publication bias . The subgroup analysis revealed that high MET GCN or protein expression was also significantly associated with poor prognosis in Asian countries , but the same tendency was not observed in the non-Asian subset . The present study was performed using univariate analysis, followed by further multivariate analysis. The results of the meta-analysis showed that high MET GCN or protein expression in NSCLC patients was associated with poor OS . This effect was also significant according to multivariate analysis, showing that the MET GCN or protein expression might be an independent prognostic factor for OS in NSCLC.The methods used to detect the MET GCN impacted the significance of these results. The combined HRs of 8 FISH (included SISH and BISH) and 3 RT-PCR studies were 1.66 (95% CI: 1.28–2.16) and 2.95 (95% CI: 0.80–10.91), respectively. We observed that FISH, instead of RT-PCR, was the most widely used technology for determining the gene copy number. In clinical practice, although real-time PCR is a simple and quick method, the results do not directly reflect cancer cells because DNA is typically isolated from whole tissue specimens that also contain normal epithelial cells, inflammatory cells, and fibroblasts. FISH is generally accepted as a better technique than RT-PCR for evaluating gene copy number because FISH can be applied to formalin-fixed paraffin-embedded tumor tissues archived for routine pathological diagnosis, thus facilitating the exclusive estimation of tumor cells. Therefore, FISH is the most widely used technique in clinical practice for the detection of gene amplification to determine therapeutic strategies, such as HER2 FISH in breast cancer. The results obtained in the present study showed that increased MET GCN, evaluated using FISH, was a predictor of worse survival in NSCLC. Due to the small number of primary studies using RT-PCR for analysis, the detection of potentially important differences was limited. Moreover, IHC was the method typically used to detect MET protein expression. IHC is the standard method for the evaluation of proteins , and there was consistency in the evaluation process among studies. The results of the present meta-analysis showed that MET overexpression was associated with worse survival.Moreover, the results of the present study demonstrated that high MET GCN or protein expression was an independent negative prognostic factor in NSCLC. However, the prognostic significance of MET GCN according to the histology of NSCLC remains unclear. Go et al Park et al in situ hybridization (SISH). Silver in situ hybridization (SISH) is a new technology for gene copy assessment, with some clinical advantages compared with FISH. First, the samples are analyzed using conventional light microscopy with preserved cell morphology based on automation. The new technology facilitates the evaluation of slides through light microscopy for the simultaneous visualization of amplified signals and cell morphology, overcoming the disadvantage of FISH where the fluorescent signals gradually fade over time. This difference might explain the observed heterogeneity.We observed a considerable degree of interstudy heterogeneity. Differences in the detection methods, types and numbers of target genes or antigens, sampling sites and times, and demographic or clinicopathologic data from the included patients, should be considered as potential sources of heterogeneity. In this study, significant heterogeneity was observed among the included studies. Although we used random- and fixed-effects models for pooling data, the source of heterogeneity remained unknown. Moreover, the sensitivity analysis did not clarify the source of the heterogeneity observed in this study. The studies by Sun et al Factors associated with immunostaining can also contribute to the observed heterogeneity. Onisuka et al Some limitations of this meta-analysis need to be discussed. First, our meta-analysis is based on data from trials whose results have been published, and we did not obtain individual patient data. Use of individual patient data may further enhance the accuracy and reduce the uncertainty of the estimates. Second, significant heterogeneity was observed among the included studies. Factors associated with variability in definitions of end point, measurements, and experimental design may contribute to the heterogeneity. Therefore, validation of the prognostic power of MET GCN or protein expression should be conducted through large multicenter prospective studies based on homogeneous populations. Third, the number of studies concerning MET and the effectiveness of therapy (such as chemotherapy or EGFR TKI treatment) was too small to perform a pooled analysis. In the present study, due to the incompleteness of clinicopathological parameters, we did not perform subgroup analyses between MET GCN and clinicopathological parameters or between protein expression and clinicopathological parameters. Fourth, negative studies are less frequently published or published with less detailed results, making these studies less assessable, potentially leading to some bias.Despite these limitations, this meta-analysis had some advantages. First, the results obtained from the random-effects model were similar to those obtained from the fixed-effects model, indicating that the statistical results were robust. Second, the results of the sensitivity analysis were not materially altered and did not draw different conclusions, indicating that the initial results were strong. Third, Egger's test did not detect publication bias, indicating that the obtained results were not biased. Moreover, the study quality scores, assessed using the Newcastle–Ottawa quality assessment scale, were >5, suggesting that the results of the present meta-analysis were convincing.In conclusion, this meta-analysis indicated that increased MET GCN and protein expression was significantly associated with poorer survival in patients with NSCLC; this information could potentially further stratify patients in clinical treatment.Table S1Assessment of Newcastle-Ottawa Scale methodological quality of cohort studies.(DOC)Click here for additional data file.Checklist S1PRISMA Checklist.(DOC)Click here for additional data file.