Random-effects meta-analyses calculating relative risks (RRs) were conducted to synthesize effect sizes of individual studies. Twenty-five studies met eligibility criteria. Use of statins was significantly associated with a reduced risk of all-caused dementia  = 0.849, 95% CI = 0.787–0.916, p = 0.000), AD , and MCI , but no meaningful effects on incident VaD . Subgroup analysis suggested that hydrophilic statins were associated with reduced risk of all-caused dementia  and possibly lower AD risk . Lipophilic statins were associated with reduced risk of AD  but not all-caused dementia . In conclusion, our meta-analysis suggests that the use of statins may reduce the risk of all-type dementia, AD, and MCI, but not of incident VaD.We conducted a systematic review and meta-analysis to investigate whether the use of statins could be associated with the risk of all-caused dementia, Alzheimer’s disease (AD), vascular dementia (VaD), and mild cognitive impairment (MCI). Major electronic databases were searched until December 27 Dementia is an age-related neurodegenerative disease, characterized by a progressive decline in cognitive function often encompassing several domains 2. According to the World Health Organization (WHO) estimates, the number of individuals affected by dementia is expected to triple by 2050, with the rapid aging of populations globally [http://www.who.int/ageing/en/]. Meanwhile, the individual, societal and healthcare costs associated with dementia are steeply increased3. Unsurprisingly, the prevention of cognitive decline and dementia is a worldwide public health issue4.As the life expectancy is getting longer worldwide, the number of people affected by cognitive decline and dementia is steadily increasing5. Statins not only lower serum cholesterol levels, but also inhibit pivotal enzymatic reactions (e.g. the isoprenylation of a subset of GTPases)6 that lead to amyloid deposition and plaque formation; both are considered cornerstone pathways underpinning the development of Alzheimer’s disease (AD)7. Epidemiological data have supported a possible association between the use of statins and the risk of dementia, but evidence appears controversial. Some observational studies have demonstrated that statins reduce the risk of dementia or incident AD17, whereas several others have failed to replicate those findings25. Several possible factors may contribute to discrepant findings across studies. First, statins might exhibit neuroprotective effects limited only to earliest stages of AD26. Some studies have shown that statins exert more robust protective effects upon cognition for subjects younger than 80 at baseline14. Second, the degree of exposure (i.e. time and dose of statins use) could influence outcomes15. Third, clinicians might not be willing to prescribe statins for patients with pre-existing poor cognitive status due to concerns about possible treatment-related side effects, and lesser benefit because of expected diminished life expectancy28. Fourth, ApoE genotype might alter the association between use of statins and incidence of dementia. One recent study has shown that statins may be more beneficial in AD patients with homozygous ApoE4 genotypes29. Finally, the effects of confounders may limit inferences from observational studies30, and hence may vary depending on both the specific population being examined and potential confounding variables that are controlled for in multivariable analysis.Recently, interest has arisen in the potential for statins to delay cognitive decline in people with older age. Statins are drugs that inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase36. A meta-analysis including case-control and cohort studies demonstrated statins use did not confer a protective effect on the risk of dementia or AD35. However, another meta-analysis of 8 prospective cohort studies found that the use of statins was associated with a significantly reduced relative risks (RRs) of dementia by 39%32. In the most recent study published in 2013, Richardson et al. found a reduced RR for all-caused dementia, AD, or MCI based on observational studies, while 16 cohort studies were available when this previous meta-analysis was conducted37. The majority of these meta-analyses have focused solely on statins use and the risk of all-caused dementia and AD. Only one meta-analysis had examined a possible effect in reducing the incidence of MCI37 and to our knowledge, no previous meta-analyses has separately assessed the risk of vascular dementia (VaD). Several new reports (including new studies which included different (i.e. Asian) populations39 have been published in more recent years41. In addition, possibly due tolimited evidence no previous meta-analysis has explored potential sources of heterogeneity across studies.Several meta-analyses of observational studies have previously been conducted to examine the potential role of statins on incident mild cognitive impairment (MCI) or dementia, but have provided conflicting results thus farTherefore, the current systematic review and meta-analysis aims to reappraise available evidence from prospective studies, which investigated whether statins use could diminish incident all-caused dementia, AD, VaD, and MCI. In addition, due to the anticipated larger current evidence base, we aimed to explore potential sources of heterogeneity across studies.Overall, 3,824 unique references were identified after database searching, while 27 were identified from other sources. Of those, 2402 were excluded after title/abstract screening because they are not related to this meta-analysis; the main target of current meta-analysis aimed to discuss the association between statins and dementia. Total 238 articles were scrutinized, and 213 were excluded with reasons , 14 studies assessed the association of statins use and incident AD , 3 studies investigated VaD , and 6 studies investigated MCI , respectively. Across those 25 studies, Supplementary material Table Among included studies, 16 studies investigated the association of statins use and incident all-caused dementia  (7.00–8.00). The score in each domain of the NOS is provided in Supplementary material Table 50, participants who received statins were significantly less likely to develop all-caused dementia compared to those who were not treated with statins  .Across 16 studies01) Fig. . There wSubgroup analysis showed the use of hydrophilic statins were associated reduced risk of all-caused dementia , whereas the use of lipophilic statins were not associated with this outcome  Fig. . Regardi51, participants who were treated with statins were significantly less likely to develop AD compared to those who were not treated with statins . There was evidence of publication bias through Egger’s test was observed  . Therefore, potential sources of heterogeneity were explored.Across 14 studies39) Fig. . We usedSubgroup analyses showed that the use of lipophilic statins was associated reduced risk of AD , while the use of hydrophilic statins reduced the risk of incident AD at the statistical trend level  Fig. . We coul23, no significant difference in the incidence of VaD was observed between participants with statins treatment and those without . There was evidence of publication bias through Egger’s regression . We used Duval and Tweedie’s trim and fill procedure toward left to adjust effect size estimates to publication bias and the results remained non-significant . No significant heterogeneity was found   Fig. .48, participants who were using statins were significantly less likely to develop MCI compared with those who did not receive statins . There was evidence of publication bias as indicated by Egger’s regression . We used Duval and Tweedie’s trim and fill procedure toward the right to adjust effect size estimates to publication bias and the results tend to insignificance . Additionally, there was significant heterogeneity was found . Therefore, potential sources of heterogeneity were explored.Across 6 studiesIn meta-regression analyses, the percentage of male proportion  and number of covariables  emerged as significant moderators of outcomes , myopathy , acute renal failure , and cataract .None of studies, except one52 allowed a more accurate exploration of potential sources of heterogeneity.The current meta-analysis of 25 cohort studies provides the most comprehensive meta-analytic evidence regarding the effects of statins on incident all-caused dementia, AD, VaD, and MCI. We found that statins users, without baseline cognitive dysfunction, had a significantly reduced risk of developing all-caused dementia, AD, and MCI. Statins use was associated with a 15.1%, 28.1%, and 26.3% lowered risk of developing all-caused dementia, AD, and MCI respectively. Nevertheless, the few studies available provided no evidence that statins use could prevent the onset of VaD. In addition, the larger body of data included in the current meta-analysis compared to previous similar efforts53. Besides, both our study  and previous work by Richardson et al. found the statins appear to provide the stronger protective effects for incident MCI . However, compared to the Richardson study, we added two studies and enrolled more subjects in the present meta-analysis45. Nonetheless, the evidence base appears to consistently indicate that statins may protect against early stage cognitive decline, such as MCI36. A longitudinal study of 6,600 subjects with normal cognition or MCI even showed statins exert a cognitive protection among those with normal cognition at baseline but not MCI, indicating statins might exert benefit before MCI status54.The current meta-analysis suggests that statins may offer a more significant preventative benefit for neurodegenerative dementing illness such as AD than for all-caused dementia55. We hypothesized that statins may reduce the development of VaD via alleviating or preventing cerebrovascular disease, which are the main risk factors for VaD58. However, the negative findings between VaD and statins from this meta-analysis might be due to the few number of included studies. Nevertheless, we considered adjust relative risks of individual studies. Therefore, it is possible that statins might not offer an independent preventative benefit for VaD. Besides, these studies did not account for important factors (such as plasma lipids or apolipoprotein E genotype)23, which might influence the results. Thus, clearly more research is required to consider if statins may confer a protective effect over future VaD.Importantly, this is the first meta-analysis to examine the association between statins use and risk of VaD, although no significant differences emerged . Atherosclerosis is considered to be responsible for diffuse periventricular white matter abnormalities, which are one of major pathophysiological mechanisms of VaD59. Besides, statins in preclinical models reduced brain cholesterol levels, leading to lower neurofibrillary tangles60. These effects could result in lower risk of dementia. The meta-regression analysis of present study could support this hypothesis, showing statins provide higher cognitive protective effect in studies with higher percentage of cholesterol >200 mg/dl . Second, statins may exert anti-inflammatory effects in the brain. For example, statins significantly reduced β-amyloid-induced production of pro-inflammatory cytokines, such as interleukin (IL)-1beta, IL-6 and tumor necrosis factors-gamma (TNF-gamma) in the hippocampus61. This could be a relevant effect since accumulating evidence indicating that both peripheral immune activation and neuroinflammation are involved in the pathophysiology of AD. Third, statins exert cholesterol-independent effects , that showed antiproliferative and antithrombotic benefits and improved endothelial dysfunction, which plays import role in the initiation of inflammatory process-atherosclerosis62. Taken together, statins could attenuate the cognitive decline via, at least in partial, decreasing anti-inflammatory action, attenuate formation of beta-amyloid (β-amyloid) and neurofibrillary by lowering cholesterol levels, and pleiotropic effects.The exact mechanism of statins on cognitive protection in older adults has not been elucidated but it might include pleiotropic effects on several mechanistic pathways. First, in animal models, statins could decrease cholesterol levels and attenuate formation of beta-amyloid (β-amyloid), which is main component of amyloid plaques in the brains of individuals with AD38 but other showing no difference between hydrophilic and lipophilic statins10. In the current meta-analysis, hydrophilic and lipophilic statins significantly decreased the incidence of all-caused dementia and AD, respectively. Studies had shown the lipophilic statins can more easily than hydrophilic statins cross the blood-brain barrier, thus potentially providing more robust benefits for the prevention of neurodegenerative disease64. Our results suggest that hydrophilic statins could exhibit more extensive protective effects than lipophilic statins in preventing all-caused dementia and possibly AD. However, these results should be interpreted cautiously. It is noteworthy that both forest plots of hydrophilic and lipophilic statins tended to the left, which means favor statins‘ preventive effect on all-caused dementia and AD. Although some results such as association between lipophilic statins and all-caused dementia and between hydrophilic statins and AD did not show significant differences, these make us not to reject the null hypothesis. Additionally, two recent epidemiological studies without non-statins comparator groups and therefore did not meet eligibility criteria for this study warrant mentioning. Zissimopoulos et al. used administrative claims data from Medicare beneficiaries to analyze the association between statin use with AD onset. They found lipophilic (simvastatin and atorvastatin) and hydrophilic (pravastatin and rosuvastatin) generally were associated with reduced risk of AD depend on different races and sex65. In contrast, Sinyavskaya et al. reported lipophic statins  were not associated with decreased incident of AD compared to hydrophilic statins , using data from the UK Clinical Practice Research Datalink cohort of patients aged 60 to 95 years who were followed for a median of 5.9 years66. Therefore, the association of statins and AD and all-caused dementia might vary across different properties of statins, sex, and race/ethnicity. Future intervention studies are needed to better study the differential effects of hydrophilic and lipophilic statins on preventing incidence of dementia or otherwise as novel neuroprotector agents for MCI and AD.Our subgroup analyses show that significant reduced risks of all-caused dementia and marginal significances of lower risk of AD were found for hydrophilic statins. In addition, lipophilic statins could reduce risks of AD but not all-caused dementia. The protective effects of statins on preventing the onset of dementia which can emerge as a function of their pharmacokinetic properties remains under debate, with some studies favoring lipophilic statins67. Caucasian subjects tend to have lower plasma exposure to and its metabolites compared non-Caucasian subjects in several types of statins69, leading to lesser treatment effect. Nearly all of recruited studies are from Western countries. The sample sizes for other ethnicities are smaller, and the estimates might be less precisely measured. Future studies to address neuroprotective effects of statins in difference ethnicities are needed. In addition, apoE4 allele is a major genetic risk factor for both all-caused dementia and AD70. It is reasonable that apoE might alter the effects of statin on the cognition as apoE plays a critical role in the transport of cholesterol and fats to the brain71. Indeed, we did find significant negative association between presence of apoE4 and AD , although not in all-caused dementia.The meta-regression analysis showed several variables were associated with statins use and the relationship with the risk of all-caused dementia, AD and MCI. One of interesting findings was statins expressed lesser cognitive preventive effect in studies with higher percentage of white ethnicity in both all-caused dementia  and AD . Pharmacokinetic differences exist among different races72 and physical exercise73 might relate to the development of dementia. To determine the intensity of social involvement is challenge and lack of objective measures in the past. Currently, there is an increasing number of commercially available devices (wearable activity trackers), which provide researchers with secure and precise means to collect, and analysis data generated by health devices74. This wearable technology could be integrated into the future cohort studies to off real-time monitoring to improve dementia prevention and more person-centered care.Some important cofounders should be considered. For example, studies have shown that different levels of social relationship76. Nevertheless, it would be potentially more informative to analysis all studies with investigating the effect of statins on the cognitive function change measured by objective tools. Future studies could be conducted to address this issue. Third, we only included peer-reviewed articles published in English language and thus it is possible that we might have missed some non-English studies. However, no publication bias was evident for almost all our outcomes. Fourth, meta-regression could not be performed for studies of VaD and MCI due to few datasets available. Fifth, none of the included studies used a propensity score or a matching criterion between statins users or not and so another potential bias could be present in our findings. In an attempt, to provide a better control of potential confounders we also performed meta-analyses considering the most fully adjusted outcome measures of individual studies. Finally, several diagnostic criteria for dementia  as well as different methods to assess the use of statins  across included studies may also limit the comparability and synthesis of studies included in this meta-analytic review.Several limitations should be addressed. First, the study is a pooled synthesis of observational studies which are susceptible to the effects of chance, bias and confounding and thus, results should be interpreted cautiously. Second, we excluded studies that considered cognitive function change as endpoint and did not discuss the clinical trajectory at every clinical assessment point until the last assessment. However, this is beyond the scope of current meta-analysis. Although the U.S. Food and Drug Administration (FDA) issued a statement regarding poor cognitive status associated with statin use in 2012, no convincing evidence so far support this warningThe use of statins appears to have beneficial effects in reducing incidence of all-caused dementia, AD, and MCI, but not VaD. Subgroup analyses suggest that hydrophilic and lipophilic statins showed a beneficial effect in preventing all-caused dementia and AD, respectively. Thus, our data suggest that the use of statins may confer a potential benefit for the prevention of dementia.77  guidelinesth, 2017 78, National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA)79, diagnosis of MCI was based on Petersen’s, International Working Group, or National Institute on Aging-Alzheimer’s Association workgroups diagnostic systems81; (4) with a follow-up period longer than one year and (5) peer-reviewed article written in English language.The following eligibility criteria were applied: (1) prospective cohort studies investigating the effects of statins vs. participants not taking statins; (2) participants had to be cognitively healthy at baseline and without a history of cognitive dysfunction; (3) outcome measures had to include either incident all-caused dementia or otherwise specific-caused dementia (i.e. AD or VaD) or mild cognitive impairment (MCI) based on Diagnostic and Statistical Manual of Mental Disorders (DSM)The exclusion criteria were: (1) cross-sectional studies; (2) retrospective case-control studies; (3) clinical trials ); and (4) studies that did not assess incident dementia or MCI at endpoint; (5) conference abstracts and studies published in languages other than English.82. For prospective cohort studies, the NOS considers three domains: selection of participants (maximum of four stars), comparability of groups (maximum of two stars), and measurement (maximum of three stars). Scores were ranged from 0 (the lowest) to 9 (the highest), and a score higher or equal to 7 indicated high methodological quality.We used the Newcastle-Ottawa Scale (NOS) to rate the methodological quality of included studiesThe primary outcomes were risk of all-caused dementia, AD, VaD, and MCI considering statins use as the exposure. Pooled estimates between statins use and dementia were calculated by using fully adjusted RRs (aRRs) provide in each publication .50, DSM-IV51, NINCDS-ADRDA51, ICD-940, or ICD-10 50; some studies did not provide the criteria for interested outcome47; diagnosis of MCI was defined based on Petersen criteria42, probable prodromal AD16 or decline of specific cognitive decline  word list)48.A pre-specified data extraction form was used to extract data for this meta-analysis. Data was extracted for each study included basic characteristics of participants , study duration , substance use , prevalence of co-occurring medical conditions , percentage of > or = 1 Apolipoprotein E epsilon 4 (ApoE4) (ApoE4 carrier), cholesterol > 200 mg/dl and percentage of overweight/obese people ). Diagnosis of all-caused dementia, AD, and VaD were based on DSM-III-RWhen data were not available in the articles, we electronically contacted the authors in at least two separate occasions to provide additional data.2 metric83. We changed ORs to RRs if data available in three recruited studies51 using the formula suggested by Cochrane Hanbook84. Therefore, the effect size (ES) and its 95% confidence interval [CI] was defined as RRs to indicate the difference of the incident rate of all-caused dementia  as a function of statins use (exposure). A similar analysis was undertaken pooling the RR of each study, adjusted for the highest number of potential confounders available.All meta-analytic procedures were performed with the Comprehensive Meta-Analysis software, version 3  software. For each meta-analysis, we considered the risk measure of each study which was most fully adjusted for potential confounders. We conducted the meta-analyses with random-effects models due to the anticipated heterogeneity across studies. Heterogeneity was assessed with the Cochran Q test and the I38. Atovastatin, fluvastatin, pravastatin, and rosuvastatin are hydrophilic statins, whereas cerivastatin, lovastatin, and simvastatin are more lipophilic statins85. Besides, status of statins usage (current and former use) was undertaken. Subgroup analyses were performed when data from at least three datasets.Subgroup analyses stratified by pharmacokinetic properties of statins (hydrophilic vs lipophilic), as previous studies, showed inconsistent results regarding the protective effect of hydrophilic and lipophilic statins on cognition86. Additionally, to account for publication bias, we used the trim-and-fill method, based on the assumption that the effect sizes of all the studies were normally distributed around the center of a funnel plot; in the event of asymmetry, this method adjusts for the potential effect of unpublished (trimmed) studies86. In addition, we conducted a meta-regression analysis using an unrestricted maximum likelihood method to explore potential moderators. The following variables were considered for meta-regression analyses: mean age, male gender (%), education (years), percentage of ethnicity, study duration , and prevalence comorbidities , percentage of presence of ApoE4 (ApoE4 carriers), total NOS scores, BMI > 25, cholesterol > 200 mg/dl and numbers of covariates adjusted. The meta-regression procedure was only undertaken when moderator variables were available from more than 5 individual studies. Statistical significance was set as two-tailed P value less than 0.05.Publication bias was assessed via a visual inspection of funnel plots and by means of the Egger’s regression testSupplementary Online Materials


Klebsiella strain was regarded as responsible for treatment failure. Intravenously administered imipenem during the sixth week led to sustained resolution of fever. Resource-limited hospitals can incur prohibitive costs from ESBL-producer infections because of diagnostic limitations and consequent treatment failure involving prolonged supportive therapy.We report on an 8-year-old patient with septicaemia unresponsive to therapy for five weeks. Undetected, extended-spectrum β-lactamase (ESBL) production by the infecting  A 2006 report of the Infectious Diseases Society of America listed ESBL-producing Klebsiella pneumoniae and Escherichia coli among drug-resistant microbes for which new therapies are urgently needed.2 Reports show that the ESBL problem is rapidly evolving and increasing in severity and scope with the discovery of new ESBLs, particularly the CTX-M types, which have become the most prevalent.3 The β-lactamases are the greatest threat to the usefulness of β-lactam antibiotics such as the penicillins and cephalosporins. Of all the different types of β-lactamases, ESBLs currently have the greatest clinical impact in terms of diversity and distribution as well as the ability to hydrolyse expanded-spectrum third generation cephalosporins. The earliest variants of ESBLs originated as a result of point mutations in the genes for broad-spectrum β-lactamases whilst newer ones, including the most successful such as CTX-M, arose by acquisition from the environmental metagenome through horizontal gene transfer.4 Cephalosporins as bactericidal, cell wall-active β-lactam agents were introduced in the 1980s and as a result of effectiveness against broad-spectrum β-lactamases became standard for treatment of severe conditions such as bloodstream infections, pneumonia and intra-abdominal infections, until ESBLs started compromising usefulness in response to overuse and selective pressure. Organisms that produce ESBLs are an important reason for therapy failure with cephalosporins and have serious consequences for infection control. Furthermore, CTX-M ESBL enzymes have been associated with coresistance to other agents including trimethoprim-sulphamethoxazole, tetracycline, gentamicin, tobramycin and ciprofloxacin.5 It is essential that clinical microbiology laboratories rapidly and reliably detect and report ESBL-producing organisms.The first report of plasmid-encoded β-lactamase capable of hydrolysing the extended-spectrum cephalosporins was published in 1983.An eight-year-old girl presented with a two-week history of fever, abdominal pain, passage of watery stool and recurrent vomiting. There was also a history of frequent micturition with occasional dysuria but neither haematuria nor passage of dark-coloured urine. Prior to presentation in the teaching hospital, she had been admitted to a distant private hospital for five days, where she was treated with amoxicillin, ciprofloxacin and artesunate (doses and duration of treatment are unknown). She was discharged from that hospital because her state of health was not improving significantly and also because of the need for better family support. There was no other history of hospital admission or blood transfusion and no history suggestive of haemoglobinopathy. Immunisation and nutritional history were essentially normal.General physical examination revealed a conscious but ill-looking, somewhat pale, febrile (temperature 38.5 °C) girl. She was moderately dehydrated and jaundiced. Her weight was 22 kg (86% of expected weight for age). There was no facial or pedal oedema. The respiratory rate was 32 cycles per min and breathing was regular; pulse regular at 120 beats per min and with good volume. Her blood pressure was 90/50 mmHg. The significant systemic findings on examination at admission were severe suprapubic tenderness, moderate hepatosplenomegaly  and negative renal angle tenderness. All other systems were normal.Klebsiella sp. on three occassions. Once, the isolate was sensitive to gentamicin and ceftriaxone but resistant to all available antibiotics tested on the other two occasions. Urine and stool cultures did not yield growth of any pathogens. Screening for human immunodeficiency virus (HIV), hepatitis C virus (HCV) and hepatitis B surface antigen (HBsAg) was negative.Conventional blood cultures were done at five different times after admission. There was a growth of 9/L – 9.2 x 109/L and an essentially normal platelet count (260 x 109/L). The erythrocyte sedimentation rate was 80 mm/hr (Westergreen method) and the haemoglobin phenotype (by electrophoresis) was AS. Serum biochemistry parameters were all normal except for conjugated hyperbilirubinaemia. Repeated abdominal ultrasonography showed findings that are consistent with hepatosplenomegaly in a septicaemic patient.Full blood counts showed a haematocrit ranged between 14% and 30%, white cell counts of 8 x 10Whilst in hospital and when fever was uncontrolled and persistent, the patient was given fresh whole blood transfusions thrice and exchange blood transfusions twice, amongst other forms of treatment.Klebsiella sp. isolate from the patient was tested and confirmed to be producing an ESBL. Immediately following this test result, treatment was commenced with imipenem  and there was dramatic resolution of fever. The patient remained free of fever for one week after receiving imipenem and was subsequently discharged. Two weeks later, when she reported for follow-up, she was still fever-free and healthy.Fever remained persistent for five weeks following admission, despite different courses of antibiotics involving ciprofloxacin, gentamicin, ceftazidime, ceftriaxone and amoxicillin-clavulanic acid . It was Klebsiella sp. isolate was identified as Klebsiella pneumoniae subspecies pneumoniae using the API 20E identification strips for Enterobacteriaceae . Presumptive ESBL phenotypic testing and confirmation in the organism was performed by disc diffusion tests on Mueller Hinton agar by employing ceftazidime (30 µg) and cefpodoxime (10 µg) alone and in combination with clavulanic acid as ceftazidime-clavulanic acid (30/10 µg) and cefpodoxime-clavulanic acid (10/1 µg) respectively. Results were interpreted using the Clinical and Laboratory Standards Institute (CLSI) criteria for disc diffusion.6 Antimicrobial susceptibility testing for the organism was carried out by the disc diffusion technique according to the guidelines and recommendations of CLSI.6 The isolate was resistant to streptomycin, gentamicin, chloramphenicol, tetracycline, nalidixic acid, ciprofloxacin, ampicillin, trimethoprim, sulphamethoxazole, ceftriaxone, cefepime and amoxicillin-clavulanic acid, but susceptible to imipenem.The 8 As shown in dfrA17 cassette encoding resistance to trimethoprim, and an aadA4 aminoglycoside resistance cassette.9 Since the integron did not contain an ESBL cassette, we screened the isolate for blaCTX-M type genes, employing primers that amplify an internal fragment from multiple blaCTX-M alleles (10. The resulting 550 bp product shown in blaCTX-M-15.Genomic DNA was extracted from the isolate using the Wizard genomic extraction kit (Promega) according to the manufacturer’s directions and used as template for PCR reactions targeting resistance elements and genes. Platinum PCR Supermix (Invitrogen) was used for all reactions, and PCR cycle conditions were as recorded in the original articles describing the primers . We empl alleles 10. The rThe cost of repeated investigations  was N14 11 In contrast, data on the epidemiology of ESBL enzymes is very limited in Nigeria. Molecular analysis of eight Nigerian ESBL-producing Enterobacter species in 2001 detected only TEM and SHV-like ESBLs and no CTX-M types.12 In a study of Klebsiella pneumoniae isolates associated with community-acquired urinary tract infections between 2002 and 2003 in Ibadan, Nigeria, CTX-M group 1, -like enzymes were found in 17 (57%), but CTX-M-15 was identified in only two isolates.13 Olowe et al. investigated the occurrence of CTX-M ESBL-producing E. coli and found nine of 79 ampicillin-resistant hospital isolates to be ESBL producers.14 More recently, a case of necrotising fasciitis was reported in a Nigerian patient in the UK.15Morganella morganii and Citrobacter freundii carrying the CTX-M-15 ESBL gene were isolated from the patient, highlighting the presence of CTX-M genes in Africa even though there is a scarcity of reports in the literature. Here, we describe a case of prolonged, uncontrolled fever found to be due to ESBL-producing K. pneumoniae. To the best of our knowledge, this is the first documented clinical course and outcome of ESBL-producing bacterial infection in Nigeria.The occurrence and spread of infections resulting from ESBL-producing organisms have been well documented in countries of Europe, Asia and North America.Failure to recognise and initially diagnose the presence of an ESBL-producing organism resulted in considerable expense in the management of the infection. This includes the cost of different courses of ineffective antibiotics for five weeks, exchange blood transfusions and whole blood transfusions, as well as hospital charges resulting from prolonged stay in hospital. The estimated avoidable cost of supportive therapy and investigation related to possible alternative diagnoses was almost $600 in a country where the average annual per capita income is $2300 and health care resources are severely limited.The clinical diagnostic microbiology laboratory plays a crucial part in the detection and reporting of ESBL-producing bacteria, and it is important that laboratories be fully aware of the significance of ESBL-producing organisms and the best methods for detecting them, as in our case. Resource-limited hospitals can incur prohibitive costs associated with ESBL-producer infections because of prolonged supportive therapy and treatment failure following the use of readily available antibiotics. Diagnostic improvements to allow routine detection and reporting of ESBL production in Enterobacteriaceae will help greatly in avoiding these costs.


Understanding the levels and associated factors of non-adherence to antiretroviral therapy (ART) is crucial in designing interventions to improve adherence and health outcomes of ART. We assessed non-adherence to ART among HIV-infected persons reporting ART use in a nationally representative survey in Kenya.The Kenya AIDS Indicator Survey 2012 was a population-based, household survey of persons aged 18 months-64 years conducted in 2012–2013. Self-reported information was collected on demographics, sexual behaviour, HIV status, and ART use. Blood was collected for HIV testing, and if HIV infected, CD4 and viral load testing. HIV-positive specimens were tested for the presence of antiretroviral (ARV) drugs using a qualitative ARV assay using liquid chromatography-tandem mass spectrometry. HIV-positive persons who reported receiving ART but did not have the ARV biomarker present were defined as being non-adherent to their ARV medication. We restricted our analysis to HIV-infected persons aged 15–64 years who reported receiving ART and had laboratory-confirmed results from ARV testing. Multivariate logistic regression was used to identify variables associated with non-adherence.A total of 648  tested HIV-positive of whom 559 (86.3%) had sufficient volume of blood to be tested for ARV drugs. Of those, 271  self-reported HIV-positive status during the interview and 186  of those reported taking ART. The ARV biomarker was absent in 18 of 186 individuals  who thus were defined as being non-adherent to ART. Non-adherence was associated with being aged 15–29 years  compared to aged 30–64 years, rural residence  compared with urban residence and taking recreational drugs in the past 30 days .Overall, less than 10% of Kenyans aged 15–64 years on ART were not adhering to their HIV medication, highlighting the success of the Kenyan national ART program. Our findings, however, point to the need for targeted interventions particularly for young persons, those in rural areas to improve adherence outcomes, as well as delivery of treatment programs that include psychosocial support as a preventative measure to minimize substance abuse and the risk of treatment failure. By year-end 2014, approximately 37 million people were living with the human immune-deficiency virus (HIV) globally, with nearly all from low- and middle-income countries. Of those, an estimated 15 million HIV-infected persons were receiving antiretroviral drug (ARV) therapy (ART), a doubling of numbers on ART from 2010 [According to the national HIV estimates for Kenya, 1.4 million adults were living with HIV in 2013 [High levels of adherence to ART are needed to ensure optimal benefits of viral suppression and prevention of emergence of HIV drug resistant virus . AdherenUnderstanding the prevalence of and reasons for non-adherence to ART among HIV-infected persons are important clinical and public health goals in reversing the HIV epidemic worldwide, particularly as countries move to providing ART to all persons living with HIV irrespective of CD4 levels in line with World Health Organization guidelines on ART . A literIn Kenya, published data on population-based levels of and predictors of adherence for persons taking ART are limited. Using data from the second Kenya AIDS Indicator Survey (2012), this paper describes nationally representative data on rates of non-adherence to ART among HIV-infected persons in Kenya aged 15–64 years and associations with select demographic, behavioural, and clinical characteristics.The second Kenya AIDS Indicator Survey (KAIS 2012) was a nationally representative population-based, household survey of adults and children aged 18 months to 64 years conducted from October 2012 to February 2013 The detailed methods for KAIS 2012 are described elsewhere . In brieThis analysis focuses on a subset of KAIS 2012 respondents that were HIV-infected, aged 15–64 years, self-reported that they had ever received ART and had blood specimen available for ARV drug testing. Due to the low number of children aged 18 months-14 years that were HIV-infected and receiving ART (n = 8), children were not included in this analysis.Dried capillary blood spot (DBS) and venous blood samples were transported to the National HIV Reference Laboratory in Nairobi several times a week. Dried blood spot specimens were tested for HIV using a serial testing algorithm using the Vironostika HIV-1/2 UNIF II Plus O Enzyme Immunoassay as the screening assay  and confirmed HIV antibody-positive results with the Murex HIV.1.2.O HIV Enzyme Immunoassay . Specimens testing negative by the screening assay were classified as HIV-negative. Specimens testing positive on the screening and confirmatory assays were classified as HIV-positive. Specimens with discordant results were retested using the same algorithm. Twice-discordant results were tested using polymerase chain reaction to determine the final result.CD4+ T-cell testing was conducted on HIV-positive venous blood using the BD FACs Calibur flow cytometer . HIV-positive dried blood spot specimens were also tested for HIV RNA concentration  and stored at -70C for future ARV drug testing. Virologic suppression was defined as HIV RNA concentration <1000 copies per mL.HIV-positive DBS were transported to an external laboratory at the University of Cape Town in South Africa where they were tested for the presence of ARV drugs with a qualitative ARV assay using liquid chromatography-tandem mass spectrometry. The assay, described elsewhere, has been previously applied in national household surveys in South Africa and Kenya –11. The Non-adherence to antiretroviral (ARV) drugs was the primary outcome in our analysis. To measure non-adherence to ARVs, we analysed data for HIV-infected respondents who self-reported 1) HIV-positive status; 2) that they were receiving ART at the time of the survey; and 3) had specimen available to test for an ARV drug biomarker. Individuals who reported receiving ART but did not have the ARV biomarker present were defined as being non-adherent to ARVs. A self-reported measure of non-adherence based on the question: “In the past 30 days, have you missed taking any of your ARV pills?” was also compared to the ARV biomarker results but not used to define the primary outcome of non-adherence to ARVs in this analysis. Predictor variables assessed included socio-demographic characteristics, including sex, educational level, marital status, wealth, and residence; partner and behavioral characteristics, including HIV status of partner, being sexually active, number of partners in the past year, condom use at last sex in the past year, history of transactional sex and anal sex, symptoms of sexually transmitted infections in the past year, and recent recreational drug use ; and clinical characteristics, including concomitant receipt of TB treatment, cotrimoxazole, and nutritional supplements , receipt of a HIV care box , frequency of HIV clinic visits andCD4+ T-cell count.We analysed all data using survey procedures in SAS version 9.3 . Estimates presented in these analyses were weighted to account for the sampling design and nonresponse. Frequencies and 95% confidence intervals (CI) were calculated to describe ART adherence status by select characteristics. Statistical significance in cross tabulations was assessed using the Rao-Scott chi-square test. We applied univariate logistic regression to identify potential variables associated with non-adherence to ARVs. Variables selected for univariate logistic regression represented demographic, behavioral, and clinical indicators considered to potentially increase the likelihood of non-adherence or have a higher likelihood of occurring as a result non-adherence to ART. Of note, cotrimoxazole use and anal sex were not included in the univariate model because there were zero observations for non-adherence among individuals who reported anal sex or who did not report receiving cotrimoxazole. In addition, CD4 cell count was not included the model due to the large number of missing observations. Variables with a p-value <0.20 in univariate logistic regression were entered into a multivariate model to identify independent and significant correlates of non-adherence to ART. Variables remaining in the model with a p-value <0.05 were considered statistically significant associations. Odds ratios (OR), adjusted odds ratios (AOR), and 95% confidence intervals (CI) were presented to describe the results of the regression analyses. To extrapolate the estimated number of persons aged 15–64 years on ART who were not adherent to their ARV drugs in the broader population, we applied non-normalized survey weights to the outcome variable and rounded estimates to the nearest 1,000. Non-normalized weights were based on the 2012 projected population data in the 2009 Kenya Population and Housing Census .This survey protocol was reviewed and approved by the Institutional Review Boards of the Kenya Medical Research Institute and the U.S. Centres for Disease Control and Prevention, and the Committee on Human Research of the University of California, San Francisco. All participants provided verbal informed consent and had the choice to consent separately to the interview, the blood draw, HIV testing and the storage of their specimens for future testing. Women and men aged 18 to 64 years were eligible to participate in the survey, provided they gave informed consent. For minors aged 10 to 17 years, parental or guardian consent and minor assent were both required for participation in the interview and the blood draw. Parental or guardian consent was required for children aged 18 months to 9 years to participate in the blood draw. For those less than age 18 years who were married, had children, or pregnant, they were considered as emancipated minors, using the criteria from the Kenya 2006 HIV Prevention and Control Act, and were able to provide their own consent to participate in the adult interview and blood draw. The study investigators obtained waiver of documentation of informed consent under 45 CFR 46.117 (c) for all survey participants on the grounds of minimal risk of harm to the subjects. Interviewers documented consent in each study participant’s questionnaire. Verbal informed consent with a signature of the interviewer as documentation of consent was approved by the institutional review boards.A total of 16,383 adult and adolescents aged 15–64 years were eligible for the survey, of whom 13,720 (83.7%) consented to an interview and 11,626 (84.7%) of those consented to the blood draw Among those that reported receiving ART, 9.4% (CI 4.9–13.8) did not have any ARV drugs detected and were classified as being non-adherent to their ARV drugs. Extrapolated to the population, this represented 32,000  persons aged 15–64 years on ART who were not adhering to their medication in 2012. In comparison, 14.7% (CI 8.7–20.6) self-reported that they had missed taking any of their ARV pills in the past 30 days, representing 50,000  persons on ART.The proportion of persons on ART that were non-adherent by select socio-demographic, behavioral, and clinical characteristics are shown in When considering sexual partners, levels of non-adherence to ARVs was three times higher among persons who did not have an HIV-positive partner in the past year  compared with those who had a HIV-positive partner in the past year . Additionally, persons engaging in high-risk behavior, including a history of giving money, gifts, or favors in exchange for sex  and using recreational drugs in the past month  had higher levels of non-adherence to ARV drugs than persons who had never given money, gifts, or favors in exchange for sex  and persons who had not used recreational drugs in the past month . Persons who reported being concurrently on medication to treat tuberculosis (TB) disease had non-adherence levels of 28.7% (CI 0–71.6) compared with 8.7% (CI 4.4–12.9) of persons who were not taking TB medication. A higher proportion of non-adherent persons had viral load above 1000 copies/ml,  compared to those who were adherent .In univariate logistic regression, the variables that met the criteria for inclusion in multivariate logistic regression were age (p = 0.001), education (p = 0.136), residence (p = 0.011), HIV status of partner (p = 0.135), a history of giving money, gifts, or favors in exchange for sex (p = 0.075), using recreational drugs in the past year (p = 0.021), having anal sex (p = 0.001) and concurrent TB treatment (p = 0.182) . After cUsing an objective measure of non-adherence to ART, this nationally representative population-based survey confirmed that less than 10% of HIV-infected Kenyans aged 15–64 years receiving ART or an estimated 32, 000 persons were not appropriately adhering to their HIV treatment. This low level of non-adherence to ART highlights the success of the national ART program in Kenya, where non-adherence levels are lower than reported in other sub-Saharan African countries, including: Rwanda, where 23% of ART patients were non-adherent based on 30-day recall ; Ghana, Since 2005, Kenya has implemented numerous interventions to provide treatment support and enhance ARV drug adherence among patients on ART, which have likely contributed to the high level of adherence we observed among the population of people receiving ART. These interventions include use of community and peer support systems, clinic appointment systems to enhance adherence as well as innovative use of short message service (SMS) via mobile devices , 24.Moreover, the detection of ARV biomarkers in the blood of individuals testing HIV-positive and self-reporting ARV use in our study was lower than the reported levels of self–reported non-adherence using a 30-day recall indicator. Had our results relied on self-report for defining non-adherence, population estimates of non-adherence would be overestimated by approximately 20,000 persons. Although there are limited data comparing self-reported adherence with plasma levels of ARV drugs, a study comparing the measured plasma levels of protease inhibitors with patients’ self-reported adherence found that patients who had concentrations below the assay limit of quantitation correlated with patient self-reported non-adherence in the previous day . A seconWe found correlates of non-adherence to ART, providing important epidemiologic insight on sub-populations where the national ART programme should target to improve long-term adherence and clinical outcomes on treatment. In our analysis, adolescents and young adults were more likely to be non-adherent to ART. Our findings are supported by those from a large observational study comparing adherence among adolescents and adults across 9 sub-Saharan African countries. This study found adolescents 50% times less likely to adhere to ART compared to adults . High leWe also found that ART patients who reported recreational drug use in the past year were significantly more likely to be non-adherent to their ARV drugs compared with those who did not report this drug use. Substance abuse can interfere with the ability of persons to take their ART medication as instructed. This finding is supported by other studies that have reported that the concomitant use of one or more illicit drugs with ART can negatively affect adherence to treatment , 37–39. This analysis should be considered in light of its limitations. Our estimates of non-adherence to ART relied on self-reported HIV-positive status, and if reported HIV-positive, receipt of HIV care and treatment services. Under-reporting of HIV status due to concerns around disclosure would have led to under-reporting of received HIV care and treatment services, affecting our estimates of non-adherence on ART. Because we did not collect information on length of time on ART or type of ARV medication taken, we were not able to assess whether non-adherence was affected by time on ART or specific ARV medications. Additionally, over 50% of HIV-positive venous samples were hemolyzed during transport to the centralized laboratory. However, persons without CD4+ T-cell count results were similar in age, sex, regional distribution compared with those with CD4+ T-cell count results. We therefore anticipate minimal bias in our estimates that report on CD4+ T-cell count data. The number of persons who self-reported ART use and classified as non-adherent to ARV drugs based on results from the ARV biomarker was small. Due to the small sample size, only major correlates were likely to be significantly associated with non-adherence to ART, while other potential correlates may not have been significant due to the low power to detect differences. Confidence intervals around estimates and associations are also wide; therefore, results should be interpreted cautiously. Finally, because KAIS 2012 was a cross-sectional study, it was not possible to confirm temporality and causality of associations observed.Despite these limitations, we present nationally representative data on the level of non-adherence to ART in Kenya which have not been presented elsewhere. Using an objective measure of non-adherence to medication, these data indicate that the levels of non-adherence were low, demonstrating substantial achievement of Kenya’s large HIV treatment program which had over 500,000 adults receiving ART in 2012 . To mini


This study investigated the use and safety of Magnetic Resonance Imaging (MRI) for several pathologies in patients with an implanted MRI conditional pacing system.This was a retrospective, single center study. All patients with an MRI conditional pacemaker or Internal Cardioverter Defibrillator (ICD) implanted in our center between June 2011 and March 2015 were enrolled. All MRI examinations performed in these patients were evaluated. For all patients, pacing thresholds, P- and R-wave amplitude, lead impedance and battery longevity were monitored during the entire follow-up.In total, 214 patients with an implanted MRI conditional device  were enrolled. At the time of implant, 94% of these patients suffered from a co-morbidity that may lead to a future indication for MRI. In 24 patients (11%), 36 MRI investigations were safely performed during a mean follow-up period of 406 days (51-1353 days). The majority of the scans were performed to confirm, exclude or follow-up on neurological disease (figure Since MRI conditional devices are not yet available for all patients, patients that have a high likelihood of requiring an MRI examination in the near future need to be selected before implantation of these devices.


In vitro embryo culture was performed and media from male and female blastocysts was collected into sex-specific pools. Profiling of 68 miRNAs revealed a total of eight miRNAs that were differentially expressed between female and male-conditioned media. Validation by qPCR confirmed higher expression of miR-22 (P < 0.05), miR-122 (P < 0.05), and miR-320a (P < 0.05) in female media for three additional biological replicates. To examine the potential roles of secreted miRNAs to the media in communication with the maternal environment, miR-22, miR-122, and miR-320a were each supplemented to four replicates of primary bovine endometrial epithelial cell culture. Uptake of miR-122 (P < 0.05) and miR-320a (P < 0.05) was detected, and a trend of uptake was detected for miR-22 (P > 0.05). Further, expression of the progesterone receptor transcript, a predicted target of all three miRNAs, was found to be upregulated in the cells following supplementation of miR-122 (P < 0.05) and miR-320a (P < 0.05), and a trend upregulation of the transcript was observed following miR-22 (P > 0.05) supplementation. This work demonstrates that male and female conceptuses are able to differentially secrete miRNAs at the blastocyst stage and that these miRNAs have the ability to induce a transcriptomic response when applied to maternal cells. This knowledge builds on the known dimorphic differences in conceptuses at the blastocyst stage and demonstrates a role for blastocyst-secreted miRNAs in cell–cell communication.Sexual dimorphism of bovine blastocysts has previously been observed through differences in development, cell death, metabolism, telomere length, DNA methylation, and transcriptomics. However, dimorphism in the secretion of miRNAs to culture media has not yet been evaluated. The objectives of this study were to determine if sex-specific blastocyst miRNA secretion occurs and to further investigate the role these miRNAs may have in the interaction between a blastocyst and the maternal environment.  In vitro production systems employed to generate bovine embryos, have reported that male embryos develop faster , and permission was granted by the company to perform in vitro fertilization using ovaries.This study is exempt from approval of the institutional and national requirements of Animal Care and Use Committee because cows used for Embryos and media were procured as described by 1aa culture media (1aa medium lacking FAF-BSA supplementation.Gametes were co-cultured with sperm for 20 h at which point presumptive zygotes were stripped of their cumulus cells and washed in supplemented Vigro TL-Hepes (Bioniche). The presumptive zygotes were placed 25 per drop into a 50 μL drop of CRre media  supplemeOn day 8 of development, individually cultured embryos were morphologically assessed for characteristics of the blastocyst stage of development. Embryos which developed a blastocoel cavity and demonstrated a distinct inner cell mass and outer trophectoderm were deemed blastocysts, whereas those which failed to develop to the blastocyst stage were deemed degenerate embryos. Blastocysts that were at the mid-to-expanded blastocyst stage and quality grades 1 and 2 , as well® DNA Polymerase system . Reagents [5X Green GoTaq® Reaction Buffer, dNTPs, magnesium chloride and primers . The immunology panel includes 68 immune response-related miRNAs chosen by ABCAM based on known functions related to immune response and differential regulation in plasma or serum. We hypothesized that these miRNAs could play a role in the immune response of the mother to the developing embryo. Complementary oligonucleotides which encode hydrogel microparticles were hybridized to selected miRNAs. The oligonucleotide adapter served as a universal PCR priming site, allowing for fluorescent amplification of the target. Amplified products were then re-hybridized to original oligonucleotide particles, and an EMD Millipore Guava 8HT Flow Cytometer  was used to quantify the hybridization. Measurements were performed in triplicates.For miRNA differential expression analysis between female and male embryos, a total of three pools of media were generated each for males (each pool consisted of media from 10 embryos) and females (each pool consisted of media from 9 embryos). Embryo pools were determined following IVF completion. Each pool included media from a unique set of individual embryos. Pools were designed to contain media of embryos from all three IVF runs, representing both sires equivalently across pools. RNA was extracted from each pool using a miRNeasy Serum/Plasma kit . A total of three extractions were carried out per pool, with an initial input volume of 120–140 μL media sample, consisting of an equal volume of eluent from individual embryos. miRNA profiling was performed using the Firefly® Analysis Workbench Software (ABCAM) in which miRNA expression levels of male and female embryos were compared to each other. An unpaired t-test was used to determine significant differential expression (P < 0.05) of miRNAs between male and female embryo groups.Statistical analysis of differentially expressed miRNAs was performed using the Firefly® particle technology, three miRNAs  were chosen for further analysis by qRT-PCR. A total of five additional IVF replicates were performed to generate 146 embryos derived from one sire and different dams. Fertilization, embryo grading, media collection, and embryo genotyping were performed as described above. A total of 101 blastocysts embryos were successfully genotyped for validation, resulting in 51 male and 50 female blastocysts female media - male media. The ΔCT values were evaluated for significance using an unpaired t-test for each miRNA. The TargetScan software2  and passaged using Hank’s Balanced Salt Solution, Trypsin/EDTA and Trypsin Inhibitor (Cell Applications Inc.), as directed.To investigate the potential of validated miRNAs as signaling molecules to maternal tissues, a primary cell culture system was implemented. Bovine endometrial epithelial cells (BEECs)  were cultured for 6–8 passages and seeded in a 96-well culture plate at 7000 cells per cmin vivo setting. A control of non-treated cells was simultaneously cultured. A total of four biological replicates were produced for each miRNA mimic treatment. After 24 h of co-culture with the miRNA mimic, the medium was aspirated from each well, cells were washed twice in 200 μL PBS, and lifted with trypsin. Cells were pelleted at 300 g for 5 min, and the excess supernatant was aspirated from each tube and discarded.Synthetic mimics for the miRNAs miR-22, miR-122, and miR-320a (Qiagen) — specific to those found to be differentially expressed between male and female embryos — were supplemented to BEECs to assess whether these miRNAs modulate maternal gene expression. At 24 h post-passage of cells, 50 nM mimic miRNA was added to a well of a 96-well plate containing BEECs. Notably, no transfection reagent was used in order to more closely simulate an To evaluate the effect of miRNAs on gene expression in the endometrial epithelial cells, an additional experiment was performed, in which cells were treated with 1 μL of Lipofectamine 2000  in conjunction with 50 nM of miRNA and Opti-MEM medium, as specified by the manufacturer. A control of lipofectamine-only treated cells was cultured simultaneously.T-ΔΔC method (t-test was performed to assess significance (P < 0.05).Total RNA was extracted from the BEECs using a MiRNeasy Mini Kit (Qiagen) to assess miRNA uptake by the maternal cells and expression of genes targeted by supplemented miRNAs, respectively. Reverse transcription and quantification of uptake of miRNAs in cells were performed as described for media. Reverse transcription of mRNA was carried out using an iScript cDNA kit  and qRT-PCR to evaluate the expression of candidate mRNA targets was performed using iTaq Universal SYBR Green Supermix (Bio-Rad). The β-actin gene was selected as an internal control according to its stability across samples in comparison to GAPDH. Intron-spanning primers were designed for each target mRNA to avoid amplification of genomic DNA . A total of 68 miRNAs were profiled using Firefly® technology, of which miRNAs miR-122 , miR-16 , miR-30b , miR-320a , miR-15b , miR-16-2 , miR-17 , and miR-22  were found to be upregulated in female-conditioned embryo media . A subset of miRNAs found to be differentially expressed in media of female embryos compared to male embryos  was selected for validation using qRT-PCR analysis. The qRT-PCR for miR-122, miR-22, and miR-320a was performed on three biological replicates of media for embryos produced from new dams. Expression analysis revealed upregulation of all three miRNAs in the media of females, with overall fold changes of 1.77, 1.86, and 1.86 for miR-22 (P < 0.05), miR-122 (P < 0.05), and miR-320a (P < 0.05), respectively .Out of 248 blastocysts collected through eight rounds of IVF, 158 embryos were successfully genotyped, resulting in a total of 81 males and 77 females. Thus, no significant sex bias was observed in our IVF system  and miR-320a (P < 0.05) were significantly higher in expression compared to control non-supplemented cells, with fold changes of 28.61 and 44.96, respectively. The miR-22 (P > 0.05) expression did not significantly differ between supplemented and control cells, though uptake was observed, with a fold change of 2.49.To test whether embryonic miRNAs are taken up by maternal endometrial cells, synthetic miRNA mimics corresponding to miR-22, miR-122, and miR-320a were supplemented to primary endometrial epithelial cells. 3 (PGR) gene was selected because it can be targeted by all three embryonic miRNAs, and is involved in the mediation of the effects of progesterone, a fundamental hormone for maintenance of pregnancy  and miR-320a , respectively. A trend for upregulation of expression was also seen for cells treated with miR-22 , with a fold change of 1.48. Additionally, lipofectamine-transfected cells exhibited upregulation of PGR gene expression for all three miRNA treatments, with fold changes of 1.51 (miR-22), 1.50 (miR-122), and 1.41 (miR-320a) when compared with a lipofectamine-only treated control  . SuccessInterestingly, the miRNAs investigated in this study have been shown to affect processes critical to the establishment of an interface between the embryo and the mother. Angiogenesis, a process necessary for embryo viability, is inhibited by miR-320 in rat myocardial microvascular endothelial cells . Normal PGR, and further functional analysis showed a trend of upregulation of the PGR transcript following supplementation with these miRNAs individually. Interestingly, upregulation of PGR was correlated with the level of uptake of each miRNA into endometrial cells . Most reported mechanisms for miRNA targeting of transcripts involve degradation of the transcript or inhibition of translation  collectively target nslation . Howevernslation . Particunslation . Additionslation , as wellPGR transcript by all three miRNAs demonstrates a complex collective targeting network of miRNAs working toward regulation of a common gene. TargetScan4 (PGR 3′ UTR, demonstrating the possibility that several miRNAs could also simultaneously induce effects on the PGR transcript. This gives cause for future investigation of the potential networks conceptus-produced miRNAs may form. Interpreting these interactions may better elucidate signaling mechanisms between the conceptus and mother during preimplantation development.Further, the substantial upregulation of the getScan4  analysisPGR is its interaction with progesterone, a hormone essential to the maintenance of pregnancy and thus, upregulation of PGR expression could have implications for the understanding of mechanisms of cow fertility prior to and during implantation. Establishment of uterine receptivity to the conceptus has been shown to require a loss of PGR expression in the luminal epithelium and then later from the glandular epithelium as a result of elevated progesterone (PGR in order to mediate effects of progesterone on the endometrium. Although cyclic and non-cyclic cows show little divergence in endometrial transcriptomes until day 16, termed the recognition point of bovine pregnancy (PGR in the endometrium during the preimplantation period is favorable for successful embryo development and implantation.The most well-studied aspect of esterone . Progestesterone . Studiesesterone . Embryosesterone  and artiesterone . Thereforegnancy , local ein vitro investigation of pooled media of blastocysts. In vivo models have not been employed to evaluate preimplantation embryo-secreted miRNAs. This is due partially to the challenge of developing precise techniques to measure expression changes induced by embryos within maternal tissues. For example, one study determined that transcriptomic changes occur in the epithelial cells in the oviduct as a result of embryo transfer, but individual embryo influences on the cells were undetectable and were only found to occur with the transfer of 50 embryos (Our study was based on  embryos . FurtherAlthough this study was limited to a panel of miRNAs chosen specifically for their impacts on immunity as opposed to a high-throughput sequencing approach, the results provide a basis for identifying larger-scale dimorphisms of secreted miRNAs. A comprehensive understanding of the exact roles for sexually dimorphic miRNA production has not yet been attained, but future research should focus on understanding larger-scale interactions and collective targeting of subsets of miRNAs on mRNA transcripts in order to better elucidate the roles these molecules may play in maternal cells and to accommodate optimal development of blastocysts relative to their sex.PGR is a remarkable display of the potential for miRNAs to function as signaling molecules during preimplantation development and allows for a new perspective on understanding the preparation of maternal cells for interaction with blastocysts, which already contain inherent differences due to their sex. Male and female embryo dimorphism in miRNA production allows for future discovery of invaluable biomarkers of embryo signaling, and understanding the potential roles of miRNAs in preimplantation development blastocyst-to-mother communication.The current study demonstrates the potential for blastocysts of differing sex to produce dimorphic signals in the form of miRNAs, which can collectively impose changes in transcripts of maternal cells. Endometrial cell internalization of miRNAs, and subsequent upregulation of in vitro production of embryos and editing the manuscript. HK conceived the study and participated in the design of experiments and drafting of the manuscript.NG carried out the experiments, data analysis and drafting of the manuscript. JK participated in the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Little is known about the psychobiological mechanisms of cognitive behavioural therapy for psychosis (CBTp) and which specific processes are key in predicting favourable long-term outcomes. Following theoretical models of psychosis, this proof-of-concept study investigated whether the long-term recovery path of CBTp completers can be predicted by the neural changes in threat-based social affective processing that occur during CBTp. We followed up 22 participants who had undergone a social affective processing task during functional magnetic resonance imaging along with self-report and clinician-administered symptom measures, before and after receiving CBTp. Monthly ratings of psychotic and affective symptoms were obtained retrospectively across 8 years since receiving CBTp, plus self-reported recovery at final follow-up. We investigated whether these long-term outcomes were predicted by CBTp-led changes in functional connections with dorsal prefrontal cortical and amygdala during the processing of threatening and prosocial facial affect. Although long-term psychotic symptoms were predicted by changes in prefrontal connections during prosocial facial affective processing, long-term affective symptoms were predicted by threat-related amygdalo-inferior parietal lobule connectivity. Greater increases in dorsolateral prefrontal cortex connectivity with amygdala following CBTp also predicted higher subjective ratings of recovery at long-term follow-up. These findings show that reorganisation occurring at the neural level following psychological therapy can predict the subsequent recovery path of people with psychosis across 8 years. This novel methodology shows promise for further studies with larger sample size, which are needed to better examine the sensitivity of psychobiological processes, in comparison to existing clinical measures, in predicting long-term outcomes. In psychosis, for example, a recent meta-analysis showed that both clinical and demographic variables are poor predictors of relapse, with non-significant effects observed for psychosis symptoms (either positive or negative), affective symptoms or clinician-rated insight.6 Measuring change at the level of the psychological processes that generate and maintain these symptoms may be helpful in improving the treatment evaluation as well as for predicting long-term outcomes.An important step forward in evidence-based practice, across psychiatric disorders, has been the use of objective clinical measures for the purpose of outcome monitoring in individuals, therapists and services.8 The psychological processes involved in threat are problematic to quantify by self-report measures due to subjective bias (both for patient and clinician). Functional neuroimaging has yielded robust and objective markers of threat processing in psychosis.10 Recently, there has been increasing interest in utilising these psychobiological markers to investigate the neural mechanisms of psychological therapies . In psychosis, two reports have arisen from an investigation of cognitive behavioural therapy for psychosis (CBTp) compared with treatment-as-usual.14 In the first study, we reported reduction in brain response to social threat from pre- to post- CBTp functional MRI (fMRI) measurements.13 Recently, we further showed that these activation changes were accompanied by reorganisation of numerous connections with prefrontal cortical and with several limbic brain regions.14 In line with our hypotheses, we found that connectivity between dorsolateral prefrontal cortex (DLPFC) and amygdala increased following CBTp. Under cognitive neuroscience models of emotion regulation, this could indicate an increased ability to contextualise potential social threat and thereby cognitively regulate negative affect,16 which fits with psychological treatment models of CBTp.11 An important finding was that the vast majority of connectivity changes did not correlate with symptom change, suggesting that they captured other CBTp-specific changes involved in socio-affective processing, over and above the symptom improvement captured by routine clinical measures. The two connectivity changes to correlate with improvement in psychotic symptoms were increases in DLPFC connectivity with inferior parietal lobule  and with postcentral gyrus . The IPL has previously been associated with theory of mind and cognitive insight in schizophrenia,18 and cognitive insight, which includes self-reflectiveness, has been shown to increase following CBTp,19 providing a plausible route by which changes in functional threat-related connectivity may mediate improvement in positive psychotic symptoms. An unexpected finding was that symptom improvement was also associated with DLPFC-postcentral gyrus connectivity, which may be understood in terms of its putative involvement in the mirror neuron system, specifically in somatic aspects of empathy during the processing of facial affect.20 In support of this, abnormalities in this region have been associated with deficits in emotion recognition22 and to correlate with psychotic symptoms.23 In line with this view, we found the symptom association was present for the processing of prosocial (rather than threatening) facial affect, which may be related to the assertion that paranoia may be secondary to the misperception of benign affect as threatening, for example.24Theoretical models of psychosis postulate that aberrant threat processing is key in generating and maintaining positive symptoms.14 over ~8 years since they received CBTp. Given the high variability in psychotic symptoms over time, both in terms of relapse as well as between-episode fluctuation of residual symptoms,3 we obtained monthly measurements instead of relying on a single follow-up ‘snapshot’ .In the present study, we sought to examine whether these CBTp-led changes in socio-affective processes are determinants of long-term clinical outcomes. To this end, we employed novel investigation methods to retrospectively follow-up a previously reported cohort16 Being key to affective well-being, we further predicted that this connectivity would determine subjective ratings of recovery at long-term follow-up. Finally, we predicted that the CBTp-led increases in amygdalo-IPL and DLPFC-postcentral gyrus connectivity that had previously been associated with improvement in psychotic symptoms following CBTp,14 would predict greater levels of remission in this symptom domain.We predicted that the degree to which threat-related DLPFC-amygdala connectivity increased following CBTp would predict greater long-term remission in both positive psychotic symptoms and affective symptoms, given the importance of this connection in contextualising potential social threat and in regulating affect.N=15; see 14 These participants had completed an fMRI implicit facial affective processing task and a battery of clinical measures on two occasions, pre (T1) and post (T2) receiving 6 months of CBTp. Sixteen outpatients receiving treatment-as-usual were also scanned at these time points .Participants were 22 outpatients with a confirmed diagnosis of paranoid schizophrenia , an average of 8 years (range 7–9 years) before the current study (T3). We obtained objective clinical outcomes for this entire period through case note review  as well as current subjective ratings of recovery and well-being .Consent was obtained by seeking current contact details from consultant clinicians in the services providing care for the previously recruited participants. Participants were then contacted by phone and those expressing an interest received information about the study, a consent form for accessing their electronic clinic records, self-report questionnaires assessing well-being and recovery and a prepaid envelope. The final sample of participants who consented and returned the questionnaires were reimbursed £10 for their time. Ethical approval was granted by the National Health Service research ethics committee (reference: 14/LO/0325).14 participants were presented with monochrome faces depicting fear, anger, happiness or neutral expressions,27 and had to indicate the sex of the face with a button press response. These were repeated in four blocks per condition, with counterbalancing across 16 blocks . These were additionally constrained within anatomical masks for these regions as defined by the PickAtlas toolbox.29 Significant connectivity changes following CBTp were tested by examining the interaction of group  by time (T1 vs T2). There were exclusively increases in connectivity in the CBTp group across functional connections with amygdala and DLPFC.As described in earlier reports,16 and in turn the relevance to cognitive behavioural models of positive symptoms of psychosis.8 We also included the two connectivity changes that previously correlated with improvement in positive psychotic symptoms: amygdala-IPL and DLPFC-postcentral gyrus, which had occurred for the processing of threat and prosocial facial affect, respectively.14For the present study, we focused our analyses on the change in DLPFC-amygdala connectivity that occurred during social threat processing, because of the strong theoretical link with cognitive regulation of affect26 is a clinician-administered rating of positive, negative and general psychopathology symptoms. Affective symptoms were measured from the Beck Depression Inventory, second edition.30The following clinician-administered and self-report measures had previously been administered pre- and post- CBTp (T1 and T2). The Positive and Negative Syndrome Schedule (PANSS);31), a service-user led instrument that follows theoretical models of recovery and provides a measure of constructs such as hope, empowerment, confidence, connectedness to others. This was our primary measure as it has one of the best psychometric properties of recovery measures32 and can be expected to be relatively robust to fluctuations in clinical state, making it well suited to use a cross-sectional measurement of long-term outcome. Additional measures for well-being, satisfaction and functioning were acquired . We assessed subjective recovery using the Questionnaire about the Process of Recovery , ‘partial remission’ , or ‘no remission’ .Longitudinal clinician ratings of symptoms: We retrospectively determined symptoms and functioning from electronic case note data held by local National Health Service trusts in South London. This covered the entire period between participants’ final fMRI measurements  and January 2015 (T3). Two raters followed validated operationalised criteria33 with strong associations between the ratings of symptoms made by case note ratings and PANSS in the same participants. We confirmed that reliability was also high for ratings made in the present study, with inter-rater agreement ranging from ‘moderate’ to ‘almost perfect’  of mild-to-moderate severity . ‘Moderate’ affective symptoms was rated where there was any period of distress lasting more than 3 days, where there was expression of suicidality not requiring severe management, or where there were three or more instances of ‘low’ affective symptoms present for that month. ‘Severe’ was rated for any month in which there was severe distress and suicidality requiring severe management, including hospitalisation or home treatment care. This method was shown to have high reliability and clinical validity,perfect’ .In addition to these symptom ratings, we also rated level of care needed  and occupational functioning , as a mean of validating the clinical ratings . There wMultivariate analysis of variance  was used to relate the longitudinal, month-by-month clinician ratings of psychotic and affective symptoms (T2 to T3) as well as subjective recovery (T3), to the functional connectivity changes (T1 to T2) as follows. All tests were performed one-tailed.rρ) to clarify the direction of associations.Percentage of months spent in each of the three symptom states was computed for positive psychotic symptoms  and for affective symptoms . To simplify the analyses and to reduce model over-fitting, we computed a single residualised variable for each symptom domain . This subsample did not differ in terms of response to CBT, either in terms of improvement in psychotic symptoms or in depressive symptoms, and additionally did not differ in task performance . These pP⩾0.42). There was a significant effect for the prosocial facial affect connection =7.83, corrected-P=0.03), which was driven by a positive association (rρ(15)=0.495, P=0.06; P=0.12).Neither of the threat-related connections were significant in the model for long-term positive psychotic symptoms (P=0.032), but not DLPFC-amygdala connectivity (P>0.99). This significant effect was confirmed to be a positive association (rρ(15)=0.49, P=0.06) (-P=0.24). The multivariate level effect approached significance =4.35, corrected-P=0.06).There was a significant effect specifically for the change in threat-related connectivity between amygdala and IPL =7.72, corrected- P=0.06) . There wP=0.04), which was positively associated with long-term recovery (rρ(15)=0.51, P=0.05) (P=0.82). There was no effect for the other connectivity changes (corrected-P⩾0.32) or at the multivariate level (corrected-P=0.11).There was a significant effect specifically for the change in threat-related connectivity between amygdala and DLPFC =6.54, corrected- P=0.05) . There wThis study utilised an innovative methodology that combined functional neuroimaging with monthly clinician ratings of symptoms over a substantial eight-year period. We showed that the reorganisation that occurs at the neural level following psychological therapy can predict the subsequent recovery path of people with psychosis across this entire period .20 It has been proposed that paranoia is causally linked to a tendency to misperceive benign affect as threatening, for example.24 Our finding that remission of positive psychotic symptoms (including paranoia) is determined by improvement in neural processes supporting affect recognition and empathy represents a novel psychobiological mechanism for CBTp.In this study, the sole predictor of long-term positive psychotic symptoms was the degree to which prefrontal cortical connectivity with postcentral gyrus had been promoted following CBTp, specifically for the processing of prosocial (rather than threatening) facial affect  was not randomised, we cannot rule out the possibility of a selection bias. There were no explicit biases in recruitment and the CBTp group did not show any differences in any of the clinical, demographic or neural measures included in the study.16 However, we previously reported that verbal intelligence (but not in other cognitive functions) was elevated in the CBTp completers compared with treatment-as-usual participants.43 It is possible that this conferred an advantage in terms of response to CBTp.There are several limitations to note of the present study. First, as is typical for follow-up studies, especially of this timespan, there was a high attrition rate. Although the final sample size remained adequate according to recommendations, for example,14 provided evidence that CBTp leads to substantial reorganisation of functional connectivity supporting social affective processing, relatively little of which is captured by measures of symptom change. The present findings extend this work by providing initial evidence that it is the degree to which this reorganisation takes place that determines sustained gains in the long-term recovery of people with psychosis. This justifies further work utilising this novel methodology on a larger scale.Our previous investigation


The long-term outcome of 228 patients with colorectal liver metastases (CRLM) who underwent preoperative chemotherapy followed by hepatectomy ± RFA were retrospectively analyzed. Stratified by chemotherapy response, patients were divided into responding (n=129) and non-responding groups (n=99). Patients who underwent hepatectomy-RFA had a greater number of metastases , a higher incidence of bilobar involvement  and longer chemotherapy cycles . In the responding group, the median overall survival (OS) and recurrence free survival (RFS) of hepatectomy-RFA and the hepatectomy alone subgroups were comparable . In the non-responding group, the median OS and RFS of patients treated with hepatectomy-RFA were significantly shorter . RFA was identified as the unfavorable independent factor for both OS  and RFS  in non-responsive patients. Local recurrence rate after hepatectomy-RFA was higher in the non-responding group . Non-response to preoperative chemotherapy may be a contraindication to hepatectomy-RFA in patients with CRLM. Liver is the most frequent site for metastasis from colorectal cancer, with more than 50% of patients developing hepatic metastases during the course of the disease , 2. LiveRFA, as a complement of hepatic resection, allows surgeons to ablate small lesions while resecting large ones, aiming to preserve adequate liver parenchyma. However, the therapeutic efficacy of hepatectomy-RFA in the treatment of CRLM remains controversial because of relatively high intrahepatic recurrence rate and unfavorable survival -14. It sPrevious studies have demonstrated that good long-term outcomes can be achieved by a combination of prior chemotherapy and RFA with or without liver resection in patients with unresectable CRLM -18. We pThe clinicopathologic features of the patients in the study are displayed in Table Patients who underwent hepatectomy-RFA had a greater number of total liver lesions . Bilobar distribution of liver lesions was also more common in patients who underwent hepatectomy-RFA . The median cycle of preoperative in hepatectomy-RFA cohort was six, compared with the four cycles in hepatectomy alone group (p=0.000). The proportion of patients who developed major complications were 29.0% and 11.9% after hepatectomy-RFA or hepatectomy alone, respectively (p=0.002).The median follow-up of the entire study population was 32 months. A total of 98 patients (42.9%) died during follow-up. Median survival was 35.7 months. The 1-, 3-, and 5-year OS rates were 93.3%, 50.5%, and 20.8%, respectively. According to response to preoperative chemotherapy, the included patients were divided into responding group  and non-responding group . Risk factors for decreased OS are displayed in Table For all included patients, there was no significant difference in survival rate between the patients who underwent hepatectomy-RFA and those treated by hepatectomy alone at 5 years  Figure . PreoperThe median follow-up of the responding group (129 patients) was 31.6 months. 5-year survival rate of hepatectomy-RFA and the hepatectomy alone subgroups were comparable  Figure . MultivaThe median follow-up of the non-responding group (99 patients) was 32 months. The median survival time for 27 patients who received hepatectomy-RFA was 18.5 months, significantly shorter than 34.2 months observed in those treated with hepatectomy alone (p=0.000) Figure . In multRisk factors for decreased RFS are displayed in Table In the responding group (129 patients), the median RFS of patients who underwent hepatectomy-RFA was 8.2 months, not significantly different from the 11.4 months after treatment of hepatectomy alone (P=0.623) Figure . MultivaIn the non-responding group (99 patients), all patients who received hepatectomy-RFA experienced recurrence within one year of operation, while the 3-year RFS rate for patients after hepatectomy alone was 19.7% (p=0.002) Figure . In multA separate analysis was performed in sixty-nine patients who underwent hepatectomy-RFA. The median survival of the responding group was 42.3 months, significantly longer than the 18.5 months in the non-responding group (p=0.000) Figure . A signiA total of 167 (73.2%) patients experienced recurrence, including intrahepatic, extrahepatic, and both intra- and extrahepatic recurrences in 128 (56.1%), 11 4.8%) and 28 (12.3%) patients, respectively. Differences in recurrence patterns among the treatment groups is shown in Table .8% and 2Recurrence pattern after surgery in the responding and non-responding groups is shown in Table Improvements in preoperative chemotherapy and combination hepatectomy with RFA allow radical treatment for an increased number of patients with CRLM. This study suggested that the benefits associated with this treatment modality were strongly impacted by response to chemotherapy.The median survival of the entire study population was 35.7 months in present study, inferior to the reported survival of 40.5–48.2 months after conversion therapy -21. We aObjective evaluation of hepatectomy-RFA compared to resection alone is difficult. Indeed, patients who underwent hepatectomy-RFA were characterized by greater number and more widespread distribution of liver lesions in current study. Besides, these patients seem to be less sensitive to chemotherapy (prolonged chemotherapy was required in 34.8% of patients). Recently, Imai et al used propensity score matching to overcome imbalance of background characteristics . After mRFA is usually regarded as an alternative therapeutic option with poor disease control for unresectable CRLM -24. IndeSeveral studies have demonstrated that progression while preoperative chemotherapy is not an absolute contraindication to liver resection for patients whose disease remains resectable -27. The Patients undergoing hepatectomy-RFA mostly had worse tumor biology than those undergoing hepatectomy alone. For responding group, hepatectomy-RFA can make patients with high tumor burden achieve a long-term outcome comparable to that of patients with favorable tumor biology. However, hepatectomy-RFA does not add any value for chemo-resistant patients, which may be attributed to the limited potential for postoperative systemic chemotherapy to cure residual micro-metastases left behind after surgery in this cohort of patients. Indeed, prolonged preoperative chemotherapy required for downstaging was associated with decreased RFS and OS in the responding group, suggesting significant effects of the extent of chemo-sensitivity on reducing recurrence and prolonging survival. Sasaki et al  recentlyThis study had some limitations. First, it was a retrospective study with relatively small sample size. Second, operations were performed by different surgeons, with their own criteria regarding patients selection for resection or hepatectomy-RFA. What’s more, some lesions judged to be unresectable in the past may now be treated by resection alone, with improved surgical techniques and skills, which may result in selection bias. Furthermore, only patients with no extrahepatic disease were eligible for the current study and these represent only a proportion of patients with CRLM. Last but not least, preoperative chemotherapy regimen was not standardized and this is also a major source of bias.Patients who underwent surgery for CRLM at the Department of hepatobiliary surgery, Cancer Hospital, Chinese Academy of Medical Sciences between January 1, 2004, and December 31, 2015, were identified from our prospective institutional database. In these patients, patients who met the following criteria were considered for further analysis. Inclusion criteria were: (1) histologically proven colorectal adenocarcinoma liver metastases; (2) preoperative chemotherapy was given, followed by hepatectomy with or without intraoperative RFA for curative intents. Excluion criteria were: (1) extrahepatic metastases detected on preoperative imaging or during surgery; (2) R2 resection; (3) postoperative deaths ; or (4) a history of prior hepatectomy for CRLM. In total, 228 patients met the eligibility criteria.All patients were evaluated preoperatively, including carcinoembryonic antigen (CEA) levels; abdominopelvic computed tomography (CT) and magnetic resonance imaging (MRI); and chest radiography or chest CT to determine the disease stage. Preoperative chemotherapy was mainly recommended to patients with initially unresectable liver metastases; or to patients with multiple high-risk factors: synchronous metastases, ≥4 hepatic lesions, primary tumor invading nearby tissues/organs and imaged mesenteric nodal disease. The definitions of unresectability were as follows: multiple liver metastases that required resection of more than 70 % of non-tumor liver for removal of all tumors, tumors invading all three hepatic veins, tumors invading both the left and right branches of the hepatic artery or portal vein, and unresectable extrahepatic metastases. Chemotherapy was composed of a combination of 5-fluorouracil/capecitabine and oxaliplatin/irinotecan with or without bevacizumab and cetuximab. Tumor response was assessed according to the Response Evaluation Criteria In Solid Tumors criteria  every two cycles. Complete response (CR) was defined as the disappearance of all signs of the current disease recorded from the start of the treatment. Partial response (PR) was defined as a decrease in the size of the tumor of ∼30%. Stable disease (SD) was defined as no disease progression or regression. Progressive disease (PD) was defined as an increase in the tumor size of >20% during or after treatment. All imaging studies were reviewed by at least two independent radiologists until the final conclusion was drawn. Surgery with curative intent was performed for hepatic lesions considered to be treatable by hepatecomy ± RFA, with the residue liver volume of at least 30%. The decision to undertake surgery for controversial cases was reached by consensus of a multidisciplinary team (MDT) including surgeons, oncologists and radiologists.During surgery, the peritoneal cavity was inspected to exclude previously undetected extrahepatic lesions. Manual liver palpation and intra-operative ultrasound were used to rule out occult lesions and confirm the number, size and location of the liver metastases. If all the lesions were deemed resectable, hepatectomies alone were undertaken. Neither the number nor the size of metastases excluded any patients from resection. Patients were treated with intraoperative RFA when one hepatic lesion was considered unresectable because of deep location or proximity to major vascular structures, so as to avoid extensive hepatectomy, especially for lesions less than 3 cm. The principle of surgery was to remove all detectable lesions with a tumor-free margin. After resection of the resectable metastases, intraoperative ultrasound was performed to guide placement of the RFA needle into the remaining lesions and then ablation was started. Successful ablation was defined as the complete destruction of the tumor with at least a 1-cm zone of normal liver parenchyma in real-time ultrasound. All specimens were subjected to histological evaluation to confirm the pathological diagnosis, number and size of liver lesions, and the width of the surgical margin. In case of multiple liver metastases, the diameter of the largest lesion was defined as the final size, and the closest margin was recorded. R1 resection was defined with a distance from the metastasis edge to the transection line of less than 1 mm.Postoperative complications were graded according to the Clavien system, and major complications were defined as any grade III or IV complication. After discharge, adjuvant chemotherapy was recommended to most patients.After surgery, patients were followed up at regular intervals. Serum CEA and imaging studies were performed to detect any intrahepatic recurrence or distant metastases. The first follow-up occurred one month post-surgery, with subsequent ones every 3 months for up to 2 years, and every 6 months thereafter.Continuous variables are expressed as the median (range). Continuous and categorical variables were compared by Mann-Whitney U test and Chi square test or Fisher’s exact test respectively. Survival analyses were carried out using the Kaplan-Meier method, with group comparisons by the log rank test. Multivariate models were used for OS and RFS using the Cox proportional hazard method. Clinicopathological factors were included in each model if they achieved a p < 0.1 for significance in univariate regression analysis. Use of RFA was included in each model, irrespective of statistical significance. P < 0.05 was considered to indicate statistical significance. OS was estimated from liver resection to death; RFS was from liver resection to the first documented disease recurrence. Statistical analyses were performed using the SPSS, version 22, Armonk NV, USA.This study showed that hepatectomy-RFA in patients who respond to preoperative chemotherapy is feasible and may be associated with a long-term outcome similar to that after hepatectomy alone. However, the poor results obtained by hepatectomy-RFA in patients with tumor stablization/progression suggest that non-response to preoperative chemotherapy is a contraindication to this treatment in patients with CRLM, and further chemotherapy may be a more reasonable choice for these patients. Chemotherapy response could be a useful tool for selecting patients for hepatectomy-RFA.