Local efficiency (LE) and node strength (NS) network properties showed the largest differences: 100% and 69.7% of nodes had reduced LE and NS in CP patients compared to HV. Moreover, 55.3% and 57.9% of nodes had decreased LE and NS in CI compared to CP patients, in associative multimodal areas. The classification method achieved an accuracy of 74.8–77.2% to differentiate patients from HV, and 59.9–60.8% to discriminate CI from CP patients. Structural network integrity is widely reduced and worsens as cognitive function declines. Central network properties of vulnerable nodes can be useful to classify MS patients.Brain structural network modifications in multiple sclerosis (MS) seem to be clinically relevant. The discriminative ability of those changes to identify MS patients or their cognitive status remains unknown. Therefore, this study aimed to investigate connectivity changes in MS patients related to their cognitive status, and to define an automatic classification method to classify subjects as patients and healthy volunteers (HV) or as cognitively preserved (CP) and impaired (CI) patients. We analysed structural brain connectivity in 45 HV and 188 MS patients (104 CP and 84 CI). A support vector machine with  Likewise, graph theory analyses of the structural brain network suggests that MS is associated with an imbalance in the integration and segregation of the network components3, which deteriorates the information flow among brain regions2, and affects their function4. In this sense, cognitive impairment has been related to a decrease in network efficiency, and changes in nodes and connections involving the insula, deep grey matter and regions of the frontoparietal network2. Measures of small-worldness and network segregation, such as local efficiency or clustering coefficient, can provide regional information by characterising the interactions of an individual node with its immediate neighbours5. Such network parameters may be modified by the disease and lead to cognitive dysfunction when the network collapses6.Multiple sclerosis (MS) is a chronic inflammatory and neurodegenerative disease of the central nervous system that leads to physical and cognitive disability. Using diffusion-magnetic resonance imaging (MRI), abnormalities in structural brain connectivity have been seen to be driven by demyelinating and neuroaxonal damage in patients with MS9, to depict disease evolution8, or to classify in the different clinical profiles of MS7. Among other classification techniques, Support Vector Machines (SVM10) are efficient, robust and accurate methods to classify data in diverse and heterogeneous contexts, such as those related to medical diagnosis12. Thus, the application of such methods may aid MS diagnosis and enhance our understanding of the network components that collapse when cognitive dysfunction is associated with the development of this disease. Therefore, the aim of this study was to characterise the regional changes in connectivity in patients with MS that influence their cognitive status. As such, using a supervised learning model we set out to provide an automatic classification method based on this information that is capable of distinguishing MS patients from healthy individuals, and of distinguishing cognitively impaired (CI) and cognitively preserved (CP) MS patients.Using machine learning algorithms, network characteristics have been used in preliminary studies to discriminate between patients and healthy subjects21 and 45 HV with no previous or current history of neurological or psychiatric diseases. Physical disability was evaluated in all the subjects using the Expanded Disability Status Scale (EDSS). The Ethics Committee of the Hospital Clinic of Barcelona approved the study, and all participants signed an informed consent. All procedures of this study were performed in accordance with the relevant guidelines and regulations.Patients with relapsing-remitting or secondary progressive MS, aged between 18 and 65 years, were recruited to this study at the MS Unit of the Hospital Clinic of Barcelona. We included 188 MS patients diagnosed according to the 2010 McDonald criteria22) was completed by all patients, and the z-scores were calculated using age and education-adjusted norms23. Patients were classified as CI when they had a z-score below −1.5 in two or more tests, while the remaining patients were classified as CP.The Brief Repeatable Battery of Neuropsychological tests , 3D-T2 fluid-attenuated inversion recovery (FLAIR) and diffusion-weighted imaging (DWI) sequences. The 3D-structural image had the following acquisition parameters: TR = 1800 ms; TE = 3.01 ms; TI = 900 ms; 240 sagittal slices with 0.94 mm isotropic voxel size and a 256 × 256 matrix size. The 3D-T2 FLAIR sequence parameters were: TR = 5000 ms; TE = 304 ms; TI = 1800 ms; 192 sagittal slices with 0.94 mm isotropic voxel size and a 256 × 256 matrix size. DWI was acquired with TR = 14800 ms; TE = 103 ms; 100 contiguous axial slices; 1.5 mm isotropic voxel size; a 154 × 154 matrix size; b value = 1000 s/mm2; 60 diffusion encoding directions and a single baseline image acquired at 0 s/mm2. In addition, field map images were generated to correct the distortions caused by field inhomogeneities .MRIs were acquired on a 3 T Magnetom Trio scanner  using a 32 channel phased-array head coil as previously describedhttp://www.xinapse.com/j-im-7-software/) using 3D-FLAIR images as a reference to improve the identification and delineation of the lesions. A WM lesion-filling approach24 was applied and for the network analysis, 76 structural brain labels were obtained using the Mindboogle (https://mindboggle.info/) and FSL-FIRST packages.White matter (WM) lesions were segmented in the 3D-MPRAGE space with the JIM 7 software  method to decompose two different tissue components, WM (anisotropic) and grey matter (GM)/cerebrospinal fluid (isotropic), which was followed by high-order probabilistic tractography using the MRtrix3 software package (http://www.mrtrix.org/)25. The WM and lesions mask were defined as tractography seeding16 to guarantee the streamline generation over pathological tissue. Using the anatomical constrained tractography (ACT) framework26, with the stopping criteria of a fibre orientation distribution (FOD) amplitude equal to 0.06, a set of 6 million streamlines was generated by the fibre tracking method. To minimise the number of false positives streamlines, we applied an anatomical exclusion criteria post-processing27.Pre-processing of diffusion MRI was achieved by DWI denoising, motion correction and geometrically unwrapping of the images28. Only connections present in more than 60% of the HV cohort were included in the statistical analyses29. Finally, age and gender correction was performed by regression analysis applied to each FA connectivity matrix.The parcellation scheme (76 regions) of structural images were adjusted to the native DWI space to define the nodes of the network. Structural brain networks were then represented by 76 × 76 weighted connectivity matrices based on the mean fractional anisotropy (FA) values for each connection (2850 network edges), obtained from lesional and normal appearing brain tissue. We incorporated a weighting factor for each streamline contributionhttps://igraph.org/), we obtained five node-based common graph measures30 from each participant’s FA-weighted connectivity matrix computed for each of the 76 regions from the parcellation map: node strength, the sum of the weights of all edges connected to a node; local efficiency, the average inverse shortest path length or global efficiency of a node calculated from the subgraph created by the node’s neighbours; clustering coefficient, the fraction of the triangles present around a node; betweenness centrality, the fraction of all the shortest path in the graph that pass through a node; and node degree, the total number of edges connected to a node.Using iGraph  when comparing CP patients with HVs, or CI with CP patients32.We defined two groups to build the classifiers, the first comprised of all MS patients relative to the HVs, and the second, to compare CI and CP patients. The construction of each SVM model implied three steps, the first of which involved the selection of the graph features, which aimed to reduce input dimensionality, particularly recommended when the number of features is higher than the number of samples and when most of the data are usually redundant or irrelevantThe second step consisted of instance creation and, given the imbalance in the number of subjects in each group, we built 100 instances of the SVM model. In order to achieve a balance, we performed random undersampling over the largest group. Thus, for the classification of MS patients relative to HVs, each instance included 90 random stratified observations  and 168 observations for the classification of CI relative to CP patients (84 CI and 84 CP patients). To find the optimal input parameters for the SVM, namely C and γ, a grid search was performed using growing sequences.k-fold cross-validation (with k = 10). Thus, at each iteration of the first type of classification, 81 observations were used for training and 9 for testing, while for the second type of classification, 152 observations were used for training and 16 for testing. We obtained several performance indicators for each validation test, including accuracy, sensitivity, specificity and F1-score33. These results were further analysed using other confusion matrix indicators to assess the overall performance of the classifiers.The third step was validation using www.R-project.org).The Shapiro-Wilks method was used to assess normality. Differences in demographic, clinical and neuroimaging data among the groups were studied using a chi-squared test, one-way ANOVA or Kruskal-Wallis tests, as appropriate, with a significance level set to p < 0.05. Levene’s test for equality of variances was used to assess the homoscedasticity assumption and we used a Welch correction to compare group differences when this assumption was violated. Multiple comparisons were analysed with a Tukey HSD or Dunn’s test as necessary, and all statistical analyses were performed using the R Statistical Software . Local efficiency was reduced in CP patients relative to the HVs in all the nodes studied (corrected p < 0.05), while in CP compared to CI patients local efficiency was decreased in 42 (55.3%) of the studied nodes (corrected p < 0.05). Regions involving bilateral pericalcarine, cuneus and lateral occipital cortex, and right parahippocampal, lingual and transverse temporal cortex were the regions with the largest differences between CP patients and HVs (corrected p < 0.05). On the other hand, areas associated with the pericalcarine, and the superior and inferior parietal cortex of both hemispheres, the right lateral occipital cortex, and the left parahippocampal cortex and cuneus were those with the largest differences between the two MS groups  nodes were weaker in CP patients than in the HVs (corrected p < 0.05), and 44 (57.9%) nodes in CI patients were weaker than in CP patients (corrected p < 0.05). Areas such as the nucleus accumbens, hippocampus and lingual gyrus from both hemispheres, right cuneus and pericalcarine cortex and the left parahippocampal area were the regions with the largest differences between CP and HVs (corrected p < 0.05). Finally, the nodes in which there was the largest differences between MS groups were the bilateral hippocampus, right precuneus, occipital cortex , the temporal cortex  and the bilateral isthmus of the cingulate cortex . Thus, 12 (15.8%), 10 13.2%) and 13 (17.1%) nodes were different in CP patients relative to HVs respectively, and 6 (7.9%), 8 (10.5%) and 10 (13.2%) between the CI and CP patients. Clustering coefficient and node degree were increased in CP compared to HV in 11 and 12 nodes, respectively, and between CI and CP patients in 6 and 10 regions. Moreover, 2 nodes showed higher betweenness centrality in CP compared to HV and 5 nodes in CI compared to CP patients . The accuracy achieved in classifying CI and CP patients was 59.46% using local efficiency and 60.77% using node strength , and no specific significance level limiting the values, the data demonstrated the viability of the approach adopted in a research environment, and the potential for its future translation into clinical practise as a complementary tool for subjects stratification.Network modifications can be considered as a hallmark of the disease and they may serve to identify patients with MS, or even their cognitive status. When we applied SVM techniques to assess this possibility, we achieved a maximum accuracy of 76.8% based on local efficiency. Classification measures with different accuracies to distinguish MS patients from HVs by applying graph metrics from both structural and functional networks have been described previously6. Reduced connectivity at nodes involving the parieto-occipital and medial temporal areas are the largest differences found in cognitively impaired MS patients. Most of these regions are considered brain hubs, maintaining a large number of connections across the brain and with intense metabolic demands, which increases their vulnerability to brain damage15. Hence, the loss of local integrity in these areas may be related to neuronal disorganization that drives cognitive impairment. The vulnerability of the frontal-parietal network components to neuroaxonal and demyelination damage was described in an earlier study we performed, along with its relationship to cognition in MS patients16.Patients with cognitive impairment had a larger reduction in local efficiency and node strength in nodes that were already affected. Such modifications support the vulnerability of impaired regions to damage, and the hypothesis that when such damage is more intense, cognitive deficits arise due to the collapse of the network or its inability to compensate the brain impairmentk-folding cross-validations, and the maximal accuracy was 60.77% using nodal strength to classify CI and CP MS patients. The difficulty in discriminating patients with different cognitive status using only MRI diffusion characteristics demonstrates the moderate relationship between structural connectivity data and cognition and the low specificity of those changes, that partially overlap between groups. Moreover, this situation highlights the need to take into account other important parameters that may influence the relationship between brain damage and function, such as brain plasticity or cognitive reserve17.However, the accuracy of machine learning techniques to distinguish patients based on their disability was more limited, as the mean accuracy was 51% (standard deviation 8.6) with 18. Recently, multi-shell DWI methods were shown to be capable of detecting potentially relevant network changes in early MS19. Further studies with these techniques may better characterise the brain microstructure in the context of this complex disease20. To improve the accuracy in classifying patients based on their cognitive status, future analyses may include clinical and other neuroimaging metrics from larger cohorts with stronger cognitive differences in a model that could modulate the changes in connectivity. However, this study focused on investigating the changes in network properties produced by the disease, relating them to cognition and their discriminative ability. Finally, the HV group sample size was quite small compared to the MS group, thus further studies with larger sample sizes and more advanced forms of the disease are advisable to validate our findings.One limitation of this study was that FA, the metric from which graph values were calculated is difficult to interpret and it has a limited capacity to estimate changes in the underlying microanatomy due to confounding factors, such as those derived from fibre crossing or orientation dispersionOur results showed a widespread reduction in the structural integrity of the brain network in patients with MS, even when cognitive decline is not yet established. Patients with impaired cognitive performance have worse brain connectivity, suggesting that as structural damage increases in vulnerable and eloquent regions, network collapse and cognitive deficits flourish. In addition, we demonstrate the feasibility of applying SVM methods based only on central structural network features to distinguish between HVs and MS patients. Future work should focus on improving the accuracy to distinguish the different cognitive profile of patients by adding more advanced microstructural features from diffusion MRI.Supplementary information.


Patients waiting or recovering from total joint arthroplasty (TJA) are at risk for falls which can lead to restriction of activity and negatively impact recovery. The objective of this scoping review is to critically appraise and synthesize the evidence in the reported number of falls, fear of falling, and risk factors associated with falls in older patients waiting for or recovering from TJA.Seven electronic databases were searched with no date limits and using language restriction (English). The inclusion criteria were 1) cohorts that included older adults 60+ years of age, 2) reported prevalence of falls, fear of falling, and/or risk factors for falls in patients who were waiting or recovering from TJA and 3) cross-sectional studies, cohort studies, and case control study designs. The quality assessment of selected articles was assessed using the SIGN Guidelines Checklist.Of the 866 citations identified, 12 studies met the inclusion criteria and were reviewed. Prevalence of falls in pre-operative TJA patients and post-operative TJA patients ranged from 23 to 63%, and 13 to 42%, respectively. Of those five studies that examined fear of falling, pre-operative TJA patients reported greater fear of falling than post-operative patients. Modifiable risk factors for falls included fear of falling, joint range of motion, and depression.An increased risk of falls in patients with TJA was reported both for patients waiting for and recovering from surgery. A number of modifiable risk factors were identified including fear of falling that could be targeted in fall prevention programs for TJA. Falls in older adults is a major public health problem given that it is the leading cause of injury in community dwelling older adults . OsteoarCauses of falls are typically multifaceted and can be attributed to intrinsic or extrinsic risk factors , 7. IntrFear of falling is a modifiable risk factor for falls which limits mobility for older adults , 15. SpeBecause few systematic reviews have specifically defined or examined falls in TJA , 5, 19, We conducted a scoping review using the method outlined by Arksey and O’Malley . InclusiThe search strategies were developed and implemented by a health sciences librarian for 7 electronic databases  and was completed on April 6, 2017. The search strategy included the following keyword terms and concepts: 1) arthroplasty 2) knee joint or hip joint 3) replacement 4) osteoarthritis 5) accidental falls , two in Cochrane Central Register of Controlled Trials. All articles were imported into Covidence where 335 duplicates were removed. We assessed 47 full articles, of which 12 studies were included in this review , 25–35 rThe definition of falls varied among the studies. Six studies used a traditionally accepted definition of falls , 30–33, n = 1) and failure to discuss presence of potential bias (n = 2).Based on SIGN guidelines, four studies were poor quality –28 six wThe mean ages of participants reported ranged from 63.9 ± 6.8  to 75.0 The number of falls in three prospective cohort studies , 32, 33 p < 0.001) [p < 0.01) [Fear of falling was reported in five studies , 29–32 a < 0.01) .Table 2In one study that examined risk factors for falls in both THA and TKA patients, patients with TKA had a greater number of factors associated with falls than patients with THA . Fear ofn = 282) of pre-operative patients indicated that their fall was related to the painful joint [Of the twelve reviewed articles, seven articles measured joint pain, function and stiffness using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores , 32, 33.ul joint .Two studies which examined joint range of motion (ROM) and the risk of falls, reported significant joint range differences between fallers and non-fallers , 31. PatThe association between the number of comorbid conditions and falls is not clearly delineated in the TJA literature. Three of five studies that specifically examined comorbid conditions and falls, reported no differences in number of self-reported co-morbid conditions between fallers and non-fallers before , 33 and Of the five cross-sectional studies that examined falls and depression, three , 29, 35 p = 0.02) [One cross-sectional study of 199 TJA patients reported a higher proportion of those with history of falls  took antidepressants as compared to those who had not fallen  ( = 0.02) . No stat = 0.02) . The useTwelve articles were included in this scoping review that specifically examined falls in patients with TJA. Falls occurring during the peri-operative period of TJA is an emerging area of interest, with 10 of the 12 studies published after 2010. A preponderance of articles, however, were small, single-center studies with the majority examining TKA. Although 3 studies examined TJA as a single cohort, the type of joint may impact the risk of falls. The few studies that followed patients before and after surgery, reported a higher proportion of falls before surgery than afterwards. Risk factors, such as environmental hazards and use of walking assistive devices are known extrinsic risk factors for community-dwelling older adults  and haveThe pattern of falls during the perioperative period is an understudied area which has clinical implications. Within this review, two studies reported the number of falls increased dramatically as the number of risk factors increase , 40. PatPain is a risk factor for falls in older adults with knee and back pain , 44. It Restricted knee and ankle ROM was associated with falls with TKA, in particular, knee extension and ankle plantar flexion were associated with falls, while total hip ROM and knee extension were associated with falls with THA. These findings support the current evidence in the literature, that significant restricted hip ROM , and ankle dorsiflexion were seen in fallers as compared to non-fallers . Given tFear of falling is a well known risk factor for falls in OA patients . Althougn < 74), which may not be reflective of the fall rate in this patient population. Methodological considerations of falls centred on defining what constituted a fall and the recall period. All studies used retrospective self-report of falls, which could lead to under-reporting, especially if the recall time is longer than 12 months [The primary aim of this review was to determine the scope of the literature that assessed falls, fear of falling, and risk factors for falls in older adults during the perioperative TJA period. Of the 12 articles identified, the majority of studies were of low or moderate quality. More than half of the studies included in this review had small sample sizes (2 months . Monthly2 months ; howeverA consideration with any scoping review is the inclusion of all studies regardless of methodological quality. In this case, we examined methods used for ascertaining falls and fear of falling both before and after surgery. This approach allowed us to look at the evidence over the peri-operative period to identify possible gaps in the literature. While others have done systematic reviews on falls only with TKA , 5 or duIn light of the high prevalence rate of falls in this patient population, the clinical implications indicate that falls prevention should be addressed both before and after surgery. Quality indicators for rehabilitation of TJA recommend balance and fall risk assessments before surgery with balance retraining during the recovery phase for both THA and TKA . RelativSubstantial evidence for fall prevention exists for older adults; however, relatively few studies have reported prevalence of falls and risk factors in patients with TJA. The findings from this scoping review suggest there are a number of intrinsic risk factors for falling in patients with TJA, both pre- and post-operative, yet further investigation needs to examine extrinsic risk factors. A number of modifiable intrinsic risk factors were identified including fear of falling which should be targeted in fall prevention programs for TJA. The findings not only advocate for high quality large scale investigations examining the burden of falls, but also warrant research of interventions to prevent falls in this patient population.Additional file 1. SearchStrategy_SUBMITTED.docx. Search strategies. Detailed search strategy for scoping review.Additional file 2. Table S1_SUBMITTED.pdf. Table S1. Characteristics of included studies. Extracted data from included studies.


Non-invasive techniques for retrieving ocular surface cells from babies infected by zika virus (ZIKV) during the gestational period remain to be determined.The aim of this study was to describe an optimized impression cytology method for the isolation of viable cells from Zika infected babies with and without Congenital Zika Syndrome (CZS) in satisfactory amount and quality to enable easy adoption in the field and application in the context of genomic and molecular approaches.Ocular surface samples were obtained with a hydrophilic nitrocellulose membrane (through optimized impression cytology method) from twelve babies referred to the Pediatric Service of the Antonio Pedro Hospital, Universidade Federal Fluminense (UFF), Niteroi, Rio de Janeiro, Brazil. After an authorized written informed consent from the parents, samples were collected from both eyes of 12 babies . Cells were used for microscopy analyses and evaluated for their suitability for downstream molecular applications in transcriptomic and proteomic experiments.Our optimized impression cytology protocol enabled the capture of a considerable number of viable cells. The microscopic features of the conjunctival epithelial cells were described by both direct analysis of the membrane-attached cells and analysis of cytospinned captured cells using several staining procedures. Epithelial basal, polyhedral and goblet cells were clearly identified in all groups. All cases of ZIKV infected babies showed potential morphological alterations . Molecular approaches were also performed in parallel. Genomic DNA and RNA were successfully isolated from all samples to enable the establishment of transcriptomic and proteomic studies.Our method proved to be a suitable, fast, and non-invasive tool to obtain ocular cell preparations from babies with and without Zika infection. The method yielded sufficient cells for detailed morphological and molecular analyses of samples. We discuss perspectives for the application of impression cytology in the context of ZIKV studies in basic and clinical research. Flavivirus genus first identified in Uganda – Zika forest, in 1947 (Zika virus (ZIKV) is an arbovirus of the  in 1947 . The vir in 1947 . However in 1947 . Congeni in 1947 . Studies in 1947 .Studies on CZS have predominantly involved analysis of brain regions  but studMolecular methodologies have been described to investigate the association between ZIKV and neurological impairment using induced pluripotent stem cells and embryonic stem cell lines differentiated in neuroprogenitors, neurons, glial cells and into the brain organoid structures . HoweverImpression cytology of ocular cells is a non-invasive method for external evaluation of ocular lesions . This teHere we describe, for the first time, a reliable ocular impression cytology protocol for different applications in the field of ZIKV infection. Our method consists of isolation of viable cells in satisfactory number and quality to enable application in the context of genomic and molecular approaches. OMIC technologies are high-throughput methodologies  that havAs an extension of the CNS, the eye displays similarities to the brain and spinal cord related to anatomy, functionality, response to damage, and immunology . MoreoveTwelve babies referred to the Pediatric Service of the Antonio Pedro Hospital, Universidade Federal Fluminense (UFF) were included in this study. All children have been followed by periodical ophthalmological examinations; samples were obtained with an authorized written informed consent from the parents. This study was approved by Universidade Federal Fluminense Ethics Committee and followed the tenets and guidelines of the Declaration of Helsinki.Ocular surface samples were collected from both eyes of eight babies according to the CZS diagnostic criteria .®, catalog number HAWP047S0). Each circular membrane was cut into four strips measuring 0.75 cm wide and 4.5 cm long approximately  was instilled into the eye before obtaining the ocular surface samples. The samples were collected with a sterilized 0.45 μm, 47 mm white plain hydrophilic nitrocellulose membrane . Tubes containing filter membranes were rapidly vortexed to allow release of adhered cells . Cell su1) and Q-Capture PRO 7 software .Viability of the collected cells was determined by the trypan blue exclusion test [9 μL of the suspension cells plus 1 μL of 0.4% trypan blue solution ]. Viable cells were counted in a Neubauer chamber. Images were acquired using on Axio microscope with 20x/0.35 and 40x/0.55 Zeiss A-Plan objectives  as the manufacturer’s instructions. This kit contains a mixture of fluorescent stains  which discriminates live from dead cells by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Analyses were performed on a fluorescence microscope  using U-MWB FITC/Texas red filter (488–570 nm excitation wavelengths), which allows simultaneous visualization of both markers.Additionally, the cytospin procedure was used to concentrate cells in suspension on a microscope slide. Cytocentrifuged preparations (200 μL of cell suspension/slide) were obtained in a Cytospin 4 Shandon  at 800 rpm for 5 min at room temperature and stained with LIVE/DEADTo evaluate microscopic features of the captured cells such as morphological types and possible alterations we then used another collection membrane directly stained with hematoxylin and eosin after fixation for 10 min in a fixation solution . Samples were hydrated with distilled water for 5 min, immersed in Harris’ hematoxylin for 2 min, washed with tap water for 15 min, counterstained with eosin for 30 s, washed with tap water for 5 min, and then dehydrated in 70, 80, 95, and 100% ethyl alcohol (rapid immersion for 5 s each). After these steps, samples were immersed in xylene (10 successive immersions for 5 min each) and mounted on slides cover-slipped using Entellan mounting medium . Images were acquired on Axio microscope using 20x/0.35 and 40x/0.55 Zeiss A-Plan objectives  and Q-Capture PRO 7 software .n = 3 patients for each group) were prepared in quadruplicate. For each pair of slides, one was stained with a Diff-Quik kit, as the standard procedure, and the other one with 0.5% toluidine blue O solution (Fisher Scientific) for 5 min. Cells were analyzed on a BX-60 Olympus microscope. A total of 423 cells were randomly analyzed  for a qualitative and quantitative evaluation of morphological alterations. The one-way ANOVA followed by Tukey multiple comparisons test were performed using GraphPad Prism version 6.00 for Windows .Another cytocentrifuged preparation was also stained for morphological analyses. For this, cell suspensions obtained as above were fixed in 4% paraformaldehyde and cytocentrifuged . Slides of captured cells (MECP2 gene that possess a significant role in embryonic development. The forward: 5′- GGA AAG GAC TGA AGA CCT GTA AG - 3′ and Reverse 5′- CTC CCT CCC CTC GGT GTT TG - 3′ (fragment size = 372 pb) primers were used and PCR conditions with modifications were applied according to previous report . Touchdown program was used in the Veriti 96 Applied Biosystems PCR thermal cycler and all samples were denatured at 95°C for 2 min and 30 s, followed by: 25 cycles of 94°C for 30 s, 65°C for 30 s, and 72°C for 1 min 45 s, followed by 10 cycles of 94°C for 30 s, 51°C for 30 s and 72°C for 1 min 30 s then a final extension 72°C for 5 min. For each sample, 5 μL of the final PCR product was checked by 1% agarose gel electrophoresis.To evaluate the integrity and quality of the genomic material of captured ocular surface cells, DNA was isolated according to QIAamp DNA mini kit , which is indicated for swabs, body fluids or washed cells. Thereafter, we used a pair of primers to amplify a region (exon 4) of the s report . The PCRFor transcriptomic experiments 9 μL of suspension solution  were viable immediately after collection in all groups and different epithelial and goblet cells were observed in Neubauer chamber. We detected the presence of viable cells adhered to the collection membrane (attached to the nitrocellulose fibers) 5 h after application of impression cytology .Microscopic analysis of the stained membranes , showed Morphological cell analyses were performed after cytospinning the cell suspensions which facilitated adhesion of the cells on the slides and resulted in enhanced visualization of cell features . The folMECP2 gene was detected in all samples (unpublished data).Genomic DNA was successfully isolated from all samples. The amount of genomic DNA ranged from 10 ng/μL to as much as 70 ng/μL, with good integrity, and sufficient for successful PCR reactions. A unique fragment of 372 bp correspondent to partial region of exon 4 of the  samples . We also samples . SufficiImpression cytology has been shown to be a simple and reproducible technique that can be successfully performed in preterm or term infants . HoweverApproximately 50% of children with CZS and microcephaly present serious eye diseases . FurtherThe human ocular surface, a specialized region derived from neurogenic ectoderm which includes the corneal, limbal, and conjunctival stratified epithelia, plays an essential role in ocular system  , and ZIKMECP2 gene, for instance, has been linked to Rett syndrome and Angelman syndrome, X-linkedDespite their importance, many questions about the genetic characteristics of conjunctival cells (mainly goblet cells) are poorly studied and deserve further exploration . MoreoveMECP2 gene and can be optimized for several other molecular studies involving other genes of interest investigating the ZIKV and microcephaly association.mental retardation, neonatal encephalopathy , some cases of autism and systemic lupus erythematosus . Here, wMolecular and cellular events, fundamental to embryological development, postnatal maturation, and maintenance of the ocular surface, are specifically regulated through advanced gene expression mechanisms. Several studies suggest a significant discrepancy between transcription and protein levels in specific cells, indicating that mechanisms related to the regulation of alternative splicing, transcript stability, translation efficiency, protein stability also participate intrinsically in gene expression . With thOMICs” research from ocular samples captured by impression cytology of babies with CZS. Studies of mechanisms involved in CZS in ocular cells require rapid, highly reproducible, and accurate quantification and can be successfully achieved with impression cytology. Ocular cell surface capture offers a powerful model for studying the pathways involved in ocular diseases associated with ZIKV.The strategies used here enabled clear detection for the first time of statistically significant morphological changes in cells from ZIKV-infected patients such as cytoplasmic keratinization and nuclear alterations as observed in other ocular disorders using cytology impression approach . To thisThe similarity of the eye to other parts of the central nervous system makes it a viable model for studying biological processes in health and CNS pathologies, like Congenital Zika Syndrome. The impression cytology with nitrocellulose membrane model developed and described in this study is a safe and effective method for the collection of ocular surface cells from babies. The sampling technique is easily implemented in field conditions and can be applied to morphological, molecular and “OMIC” analyses of Zika infected patients. On the basis of the evidence described in this study, ocular cell capture has substantial benefits as research tools for central nervous system disorders, include Congenital Zika Syndrome.All datasets generated for this study are included in the article/supplementary material.Study design and ethic aspects: Twelve babies referred to the Pediatric Service of the Antonio Pedro Hospital, Universidade Federal Fluminense (UFF) were included in this study. All children have been followed by periodical ophthalmological examinations; samples were obtained with an authorized written informed consent. This study was approved by the Universidade Federal Fluminense Ethics Committee and followed the tenets and guidelines of the Declaration of Helsinki.RB conceived the original idea, wrote the project study, provided funding, designed the experiments, organized the works groups , collected the respective informed and written parents’ consents, performed the sample preparation and storage, the analytical method development, data interpretation and wrote the manuscript. MS collected the impression cytology membranes. MS and CC performed the cohort selection. CC coordinates the clinical follow-up study. AP, PS, CB, and PF assisted on analytical method development and revision of the manuscript. LR-F and GP performed the proteomic method development. TS and RM performed the microscopy and morphological analyses. RM and EL assisted on analytical method development and critical revision of the manuscript. BL assisted in the experimental design and revision of the manuscript. All authors contributed in editing the manuscript and approved the final version.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.


Clinically relevant adverse drug reactions differ between men and women. The underlying physiological and pharmacological processes contributing to these differences are infrequently studied or reported. As gene expression, cellular regulatory pathways, and integrated physiological functions differ between females and males, aggregating data from combined groups of men and women obscures the ability to detect these differences. This paper summarizes how genetic sex, that is, the presence of sex chromosomes XY for male or XX for female, and the influence of sex hormones affect transporters, receptors, and enzymes involved in drug metabolism. Changing levels of sex steroids throughout life, including increases at puberty, changes with pregnancy, and decreases with age, may directly and indirectly affect drug absorption, distribution, metabolism, and elimination. The direct and indirect effects of sex steroids in the form of exogenous hormones such as those used in hormonal contraceptives, menopausal hormone treatments, transgender therapy, and over‐the‐counter performance enhancing drugs may interfere with metabolism of other pharmaceuticals, and these interactions may vary by dose, formulation, and mode of delivery  of the steroid hormones. Few drugs have sex‐specific labeling or dosing recommendations. Furthermore, there is limited literature evaluating how the circulating levels of sex steroids impact drug efficacy or adverse reactions. Such research is needed in order to improve the understanding of the impact of sex hormones on pharmacological therapies, particularly as medicine moves toward individualizing treatments. ADRadverse drug reactionsCARconstitutive androstane receptorCYPcytochrome P enzymesFDAFood and Drug AdministrationGPR30G‐coupled receptorsILG1Rinsulin‐like growth factor 1 receptorPI3Kphosphatidyl inositol 3,4,5, triphosphatePXRpregnane X receptorSRYsex determining region YSSRIsselective serotonin reuptake inhibitors1Adverse drug reactions are common and thought to be the fourth leading cause of death.vs 22.9%). Most ADR that impacted women were dose‐related and were common reactions mentioned in the product labeling. Among 59 ADR that were reported at least 50 times, 16 were reported more often in women than in men and four were more commonly reported in men than in women.In spite of problems with data reporting by sex, over the past several decades, studies have recognized that ADR are experienced by women more often than men 22.1SRY gene on the Y chromosome encodes the sex‐determining region Y (SRY) protein, which is a DNA‐binding protein that is important in the development of the testes that will ultimately secrete testosterone and lead to a male phenotype. The AR gene that encodes the androgen receptor resides on the X chromosome; therefore, any variant in AR that impacts function will be expressed as an X‐linked trait in males with one X chromosome.The presence of sex chromosomes, XX in female and XY in males, in all nucleated cells represents the most fundamental genetic difference between females and males. Genes on these chromosomes regulate specific and diverse physiological functions, in part, through modulation of genes on the autosomes.As the reproductive organs form, sex steroid hormones differing between males and females will be secreted and influence body size, organ size, tissue composition , gastric acid secretion, gastric emptying time, circulating proteins that may bind medications, renal function, immune reactivity, and drug metabolizing enzymes;2.2Traditionally, sex hormone signaling mechanisms were only thought to alter gene transcription through steroid response elements on specific genes, that is, genomic effects Figure .20 Howev2.3There are several mechanisms by which sex hormones may interact with medications and their metabolic pathways, contributing to ADR Figure .2.3.1Bioavailability of a drug may depend on mode of delivery and formulation. For example, transdermal formulations often require absorption into subcutaneous adipose tissue. Women typically have more subcutaneous adipose tissue than men, which theoretically could impact absorption.2.3.2SLCO1B1, is responsible for transport of estrogens including estrone‐3‐sulfate and estradiol 17β‐D‐glucuronide. Statin drugs are also transported by OATP1B1. Competitive inhibition of this transporter may occur when multiple substrates are present.SLCO1B1 genetic variants on the efficacy of statin treatment.SLCO1B1 c.521C allele, suggesting that competition for transporters may result in clinically significant drug‐hormone interactions.Drugs may compete for transporters into cells, thus affecting downstream metabolism or availability at the drug target and altering extracellular concentrations. For example, the OATP1B1 transporter, encoded by the gene 2.3.3CYP3A4, and other genes, and these receptors are activated by a variety of compounds, including steroid hormones.The pregnane X receptor (PXR) and constitutive androstane receptor (CAR) regulate expression of cytochrome P450s, including 2.3.4Steroid sex hormone status may also influence pathways contributing to the mechanism of action of medications; however, less is known related to these pharmacodynamic interactions. In one example, women were found to require a smaller dose of olanzapine in order to achieve 70% occupancy of the dopamine D2 receptor for medication efficacy.3Fluctuations in endogenous sex steroid hormones that occur naturally with the menstrual cycle, pregnancy, and with the transition to menopause have the potential to influence drug efficacy and ADRs.3.1Changes in endogenous hormones associated with pregnancy will influence drug efficacy and some medications may have adverse effects on fetal development. Indeed, developmental defects associated with use of thalidomide resulted in the exclusion of pregnant women from drug testing trials.Pregnancy does not simply increase the volume of blood and extracellular fluid that influence distribution and clearance of drugs, but the hormonal changes also influence enzyme activity. For example, activity of CYP1A2 is decreased during pregnancy affecting metabolism of caffeine and theophylline.3.2CYP19A1) in adipose tissue and skin becomes the predominant source of estrogen. With the decrease in estrogen, changes in other drug metabolizing enzymes are also observed. For example, CYP3A4 activity in the intestine is reduced by about 20%,With menopause, circulating estrogen decreases to about 90%. Conversion of androgens to estrogen by aromatase  may affect medications by altering metabolism; at the same time, drug metabolism pathways may impact exogenous hormones used for therapy. The genes encoding drug metabolizing enzymes and transporters are highly polymorphic, which is the basis of pharmacogenomics. Although the influence of genetic variation on menopausal physiology is not well understood, heritability estimates of age at menopause ranges from 31%‐78%. Thus, the question arises as to whether a pharmacogenomic approach, that is, studying the impact on exogenous hormones of genetic variation in genes encoding pathways of drug metabolism, could be applied to optimize menopausal hormone therapy or hormone therapy for other indications. This question is especially important to optimize hormone therapy for women who might undergo bilateral oophorectomy prior to the age of natural menopause and require menopausal hormone therapy to reduce the risk of multimorbidity of aging.SULT1A1, which encodes an enzyme that sulfates estrone, 17β‐estradiol, and 4‐methoxy‐estradiol might be associated with serum hormone levels of estrogen and menopausal symptoms. The SULT1A1 gene is polymorphic with both single nucleotide variants (SNVs) and whole gene deletions and duplications . When evaluating this gene, it is critical to consider the impact of both SNV and CNV simultaneously. Some of the SNVs decrease enzyme activity, while others impact transcription; gene duplication also leads to increased activity.SULT1A1 affected circulating levels of sulfated estrone and the ratio of sulfated estrogen to estrogen.Our group has taken a candidate gene approach to begin to evaluate how genetic variants in enzymes involved in estrogen metabolism might be related with response to estrogen therapies. One variant in SLCO1B1 encodes the transporter (OATP1B1) for many endogenous and xenobiotic compounds. In the context of this paper, this transporter transports estradiol‐17β‐glucuronide, estrone‐3‐sulfate, and statins from the blood into hepatocytes. Variants in SLCO1B1 associated with breast cancer risk and statin‐induced myopathy in postmenopausal women using oral conjugated equine estrogen with medroxyprogesterone acetate. In KEEPS, women with normal transporter activity had lower circulating levels of estrone and 17β‐estradiol sulfate than those with lower activity, while those with reduced transporter activity demonstrated a greater decrease in night sweats among women on active treatment.A second candidate gene 3.2.1CYP2C19 promoter.Exogenous hormones may be prescribed for a variety of purposes, including contraception. These exogenous hormones also impact metabolism of other medications. While the underlying mechanism of decreased CYP activity in the presence of exogenous hormones, such as those in oral contraceptives, is thought to be a competitive inhibition, there is some evidence that estradiol may downregulate CYP2C19 expression through the interaction of estrogen receptor (ER) α with a binding site in the CYP3A7*1C allele were more likely to have serum etonogestrel concentrations falling below the threshold of 90 pg/mL required for consistent ovulatory suppression. This allele results in adult expression of the fetal CYP3A7 enzyme, which is in the same family as the CYP3A4 enzyme known to metabolize estrogens. In addition, nongenetic variables of body mass index and duration of implant use also impacted estrogen levels. This example further highlights the importance of understanding individual variation in steroid hormone metabolism in ensuring efficacy of therapy.Genetic variation may also influence the concentrations of the exogenous hormones used for contraception and other indications. A study of 350 healthy, reproductive‐age women using etonogestrel implants for 12‐36 months were genotyped for 14 genes encoding proteins involved in steroid hormone‐related pathways.4Changes in outward appearance, physiology, and metabolism resulting from treatment with sex steroid hormones for clinically defined gender incongruenceFew studies have specifically addressed potential hormone‐drug interactions except for managing trans patients with sickle cell disease,5Although there are data supporting sex differences in ADRs, there are few systematic analyses of these adverse events by drug class that take into account stages of life (age), and hormonal status. Given that sex hormones may alter the pharmacokinetics and pharmacodynamics of medications, it is important to note that these hormones naturally fluctuate throughout the lifespan. Among females, hormones change at puberty, with menstrual cycles, during pregnancy, and at menopause; in addition, exogenous hormones may be used for contraception or to alleviate menopausal symptoms. Among males, hormones change at puberty as well as with aging, and exogenous hormones may be used. However, many preclinical basic pharmacology studies to define mechanisms of disease  do not identify the sex of the experimental material, age or hormonal status of the source of cells or tissues.The authors declare no financial conflicts of interest. VMM research is supported by a grant from the National Institutes of Health U54 AG 44170.AMM: Drafted and edited the manuscript and figures. ETM: Drafted and edited the manuscript. VMM: Drafted and edited the manuscript and figures.Not applicable as only previously reported data are presented in this review.All published studies involving humans contain a statement that the work was performed in accordance with review by Internal Review Boards and the Declaration of Helsinki and that participants signed consent documents. Papers reporting work conducted on experimental animals were conducted in accordance with the Animal Welfare Act.


Dermanyssus gallinae negatively impacts bird welfare and health, and interferes with egg production and quality, while emerging acaricide resistance limits control options. Fluralaner, a novel miticide for administration in drinking water, is approved for control of D. gallinae infestations. Mite sensitivity testing is relevant to gauge field isolate susceptibility to available treatments.The poultry red mite D. gallinae isolates collected during 2014 through 2016 from farms in Germany, France, Spain and Brazil, and a 2001 laboratory-maintained isolate were used for acaricide contact sensitivity testing. Tested compounds were cypermethrin, deltamethrin, phoxim, propoxur, and the recently available acaricides, spinosad and fluralaner. In each study, at least one isolate was exposed to increasing concentrations of at least one acaricide. In one study, additional testing determined the sensitivity of the 2001 isolate to fluralaner using a mite-feeding test, and of fluralaner, phoxim and spinosad using an immersion test. At least two replicates were used for each dilution. Vehicle and untreated controls were also included.Thirteen 90) values, the laboratory isolate was susceptible to fluralaner , phoxim (< 500 ppm), propoxur (< 125 ppm), and deltamethrin (500–1000 ppm). All field isolates remained sensitive to fluralaner concentrations ≤ 125 ppm. Spinosad LC90 values for laboratory and field isolates ranged between 2000–4000 ppm. For phoxim, relative to the laboratory isolate, there was reduced sensitivity of two German isolates (LC90 up to 4000 ppm) and two French isolates (> 4000 ppm). An isolate from Spain demonstrated reduced sensitivity to phoxim, propoxur and deltamethrin; an isolate from Brazil showed reduced sensitivity to propoxur and cypermethrin. Mite LC90 when exposed to fluralaner by blood feeding was < 0.1 ppm.Based on 90% mortality (LCD. gallinae infestations of poultry.Contact sensitivity testing indicated apparent resistance to at least one of phoxim, deltamethrin, cypermethrin and propoxur in 13 field isolates from Europe and Brazil. All isolates were highly susceptible to fluralaner. Fluralaner was approximately 1000 times more active by feeding than by contact. Fluralaner’s distinct mode of action and efficacy against isolates largely refractory to those acaricides, makes it a promising option for the control of  Dermanyssus gallinae  is an important ectoparasite of poultry that negatively impacts poultry health and welfare, reduces egg production and egg quality, and is a potential pathogen vector [D. gallinae seeks its hosts at night, taking blood meals that typically last for less than two hours, after which mites retreat into cracks and crevices of nest boxes or other suitable sites within the wider surroundings of a poultry housing unit [D. gallinae may be as short as 7 days [The poultry red mite De Geer, 778 is ann vector –11. An uing unit –14. At ts 7 days –17. Suchs 7 days , 7, 14.D. gallinae by effective and safe treatments has been a largely unmet need for nearly two decades [Until recently, the control of the poultry red mite has been largely dependent upon the use of sprayed acaricides, such as organophosphates, carbamates, macrocyclic lactones, pyrethroids, and spinosyns , 14. HowD. gallinae on EU poultry operations [During 2017, the isoxazoline compound fluralaner was approved for the control of erations . This aperations , 27. Fluerations .D. gallinae to fluralaner, and to assess the sensitivity of these isolates to commonly used mite control acaricides, six in vitro laboratory studies were initiated. Mite isolates were collected for testing from commercial poultry operations in Europe and Brazil. Comparator active ingredients were spinosad, phoxim, propoxur, and the pyrethroids cypermethrin and deltamethrin.To determine the sensitivity of field isolates of Across six separate studies, 12 field isolates were freshly collected between May 2014 and July 2015 from commercial layer, breeder, and pullet farms located in Germany, France and Spain, and in September 2016 one isolate was collected from a layer farm in Brazil. No chemical acaricidal treatment had been used in the sampled houses for at least two months prior to mite collection. In Studies 1 through 5, mites were brushed from typical refuge areas into plastic containers that contained filter paper to absorb humidity, and that had a lid-hole covered with air-permeable paper to preclude mite escape ; spinosad from APIN Chemicals , and cypermethrin from Tagros Chemicals . All compounds tested were of technical grade greater than 95%.in vitro contact sensitivity of each of the 13 D. gallinae field isolates and a laboratory isolate was tested in at least one of three separate laboratories: MSD Animal Health Innovation GmbH, Schwabenheim, Germany ; ZeckLab, Burgwedel, Germany , and the Agricultural Biotechnology Laboratory, State University of Western Paraná, Cascavel, PR, Brazil   for resistance testing of tick larvae . Two repIn Studies 2 to 5, actives were dissolved in 1-butanol + olive oil (DAB10) to obtain a 1% stock solution (100 mg substance/10 ml solvent). Test concentrations were prepared by further 1:2 dilutions with deionized water. Filter papers (8 × 10 cm) were coated with 800 μl of the final test solutions or with solvent only (control), and left for drying for 48 h at room temperature. Impregnated papers were folded and closed with clamps on two sides. Approximately 100 mites were placed into the opening of each filter paper which was then sealed and placed in a holding apparatus  test, mites were placed between two filter papers and 5 ml of test solution was added. Mites were immersed (between the filter papers) in fluralaner (0.031 to 4000 ppm), phoxim or spinosad solutions (each 2 to 4000 ppm). After approximately three minutes, the solution was poured off, the filter papers were dried with towelling paper and the mites were then placed onto a fresh triple folded filter paper that was sealed (as a sachet). After incubation for 48 h, at approximately 22°C and 90% RH, all mites were counted. There were two replicates for each test compound and the two controls, one containing DMSO solvent and one untreated.-7 to 101 ppm. Control mites were exposed to either DMSO solution or to blood only. After two hours feeding in darkness, engorged mites were collected, transferred into a glass tube containing a paper strip, closed with an air-permeable lid, and incubated for 24 h at approximately 22 °C and 90% RH. The test included two controls (one DMSO solvent and one untreated control), and was carried out in three replicates. Phoxim was not included in the feeding test because of its volatility.For the feeding test, between 100 and 150 mites were transferred into plastic vials (diameter 20 mm), each closed with a membrane (Parafilm®) that had been treated with mite-attractant solution prepared from an ethanol-butyric acid extraction of a mixture containing minced chicken skin and feathers. The vials were then placed into a glass dish containing 2 ml of a warmed blood solution comprised of a mixture of defibrinized sheep blood and stock solution, and fluralaner at concentrations ranging from 10The MM, expressed as a percentage and calculated for each test concentration approximately 48 h after contact exposure (studies 1 to 6), or 24 h after feeding (study 1 only), was calculated as follows:dead/ncounted) × 100Mite mortality =  of dead mites per test concentration; ncounted is the arithmetic mean  of counted mites per test concentration.where nThe per cent MM was calculated using the formula:C is the per cent MM of mites exposed to the control solution and MMT is the mortality of mites exposed to the test concentration. For Studies 1 and 6, a mean of the solvent and the untreated control was used as MMC.where MM90) were calculated using probit analysis (SAS® release 9.2). In Studies 2 to 5 the low number of tested concentrations limited LC90 estimations to a certain range. Results therefore focus on concentrations where mortalities exceeded 90%, with comparisons to LC90 values calculated in Study 1.In Study 1 the 90% lethal concentrations , spinosad (2000–4000 ppm), phoxim (2.6 and < 500 ppm), propoxur (< 125 ppm), and deltamethrin (500–1000 ppm) , there was an apparent decreased contact sensitivity of two German field isolates for which LC90 had increased to between 1000–2000 ppm (field isolates 10 and 11), and one isolate for which the LC90 lay between 2000–4000 ppm (field isolate 4) , MM at the maximum tested phoxim concentration (1000 ppm) was 6.9%. This isolate also showed reduced sensitivity to both propoxur and deltamethrin. Field isolate 3 from Spain also demonstrated similar reduced sensitivity to phoxim, propoxur and deltamethrin. Reduced sensitivity was demonstrated by field isolate 13 from Brazil to propoxur and cypermethrin: contact exposure to 250 ppm resulted in mortality of 15.6 and 4.1%, respectively, only marginally increasing to 19.8% and 15.6% at 1,000 ppm, the highest tested exposure  Tables ; Fig. 6.50 and LC90, MM from exposure to fluralaner was greater in the feeding test than in either of the contact tests (filter paper and immersion)  Table .Fig. 7PeD.gallinae resistance to many acaricidal classes  is widespread, and is a contributing factor to the increased prevalence of poultry red mites [90 values described in this report provide further evidence that poultry red mite resistance to multiple chemical groups has developed under commercial production conditions. From the 11 field isolates originating from layer and breeder farms in France, Spain and Germany, six had a LC90 for phoxim exceeding the recommended concentration for commercial use. Deltamethrin and cypermethrin were tested on one Brazilian and three EU field isolates, and all had LC90 values exceeding the commercial product label rates of 250 and 100–200 ppm [90 of the three isolates from Europe and Brazil exceeded 1000 ppm, a concentration that, in the authors experience, was used in poultry prior to its withdrawal from the European market.Multiple reports from individual countries have indicated that ed mites , 24, 30.–200 ppm , 32. TheD. gallinae isolates. The greatest activity of fluralaner was observed in the feeding test at extremely low lethal concentrations (LC90 < 0.1 ppm), approximately 1000 times lower than that obtained during the filter paper contact test. The latter test thus provides a “worst case scenario” with reference to fluralaner’s systemic mode of action, especially when compared to results of other acaricides with a contact mode of action. The value of more potent systemic than contact activity for fluralaner is that the innovative mode of administration (in drinking water) enables the use of a lower and more precise amount of active, when compared with a spray application.In this report, three methods were used: a filter paper test corresponding to the spray application used for the traditional treatments which are contact acaricides; an immersion test; and a feeding test which is relevant to fluralaner because mites are exposed while feeding on treated birds. Irrespective of the test method, fluralaner was consistently active at low concentrations against all the tested 90 of up to 4000 ppm for the laboratory and EU field isolates with MM assessed after 48 hours, indicates that this chemical is less potent than the other tested acaricides, including fluralaner, and is consistent with previous sensitivity reports [The spinosad LC reports . These c reports .The present studies assessed MM following exposure of a 2001 isolate, and isolates from 2014 to 2015, to phoxim, propoxur and spinosad. Using the same method at the same laboratory, the results show a declining sensitivity of field isolates to phoxim and propoxur. No change was observed for spinosad which was only launched in 2012. This supports the belief that resistance to older compounds has continued to build during the last decade .Ornithonyssus sylviarum, the northern fowl mite, particularly prevalent in North and Latin America, at similar exposure rates as those to which D. gallinae is sensitive [Results from the studies described in this report show that fluralaner is active against poultry red mites, isolated from commercial operations, showing decreased susceptibility to the most commonly used acaricides. These findings are consistent with reports of fluralaner efficacy against ensitive . They alensitive .D. gallinae is highly susceptible to the novel poultry acaricide fluralaner, with much greater sensitivity shown by exposure through feeding on blood than by contact or immersion. This differential sensitivity toward exposure through feeding aligns with fluralaner’s oral administration for systemic activity against mites. Similar susceptibility to fluralaner was observed for European and Brazilian field isolates showing apparent resistance to phoxim, deltamethrin, cypermethrin and propoxur. The different mode of action than that of other poultry acaricides, and the efficacy demonstrated against isolates largely refractory to those acaricides, makes fluralaner a promising option for the control of D. gallinae, helping to reduce or avoid the costly effects of this damaging parasite.The studies reported in this paper demonstrate that the poultry red mite


Meleagris gallopavo silvestris) across the state of Mississippi, United States. Of the total locations, 453 locations separated by a distance of ≥2 km were used to train the three ML algorithms on the OD, RD, and PC data, respectively. The remaining 210 locations were used to validate the trained ML models to measure ML performance. Three ML models had excellent performance on the RD and PC data. MaxEnt and SVMs had good performance on the OD data, indicating the adequacy of regularization of the default setting for multicollinearity. Weak learning of RFs through bagging appeared to alleviate multicollinearity and resulted in excellent performance on the OD data. Regularization of ML algorithms may help exploratory studies of the effects of environmental factors on the spatial distribution and habitat suitability of wildlife.Species distribution modeling often involves high‐dimensional environmental data. Large amounts of data and multicollinearity among covariates impose challenges to statistical models in variable selection for reliable inferences of the effects of environmental factors on the spatial distribution of species. Few studies have evaluated and compared the performance of multiple machine learning (ML) models in handling multicollinearity. Here, we assessed the effectiveness of removal of correlated covariates and regularization to cope with multicollinearity in ML models for habitat suitability. Three machine learning algorithms maximum entropy (MaxEnt), random forests (RFs), and support vector machines (SVMs) were applied to the original data (OD) of 27 landscape variables, reduced data (RD) with 14 highly correlated covariates being removed, and 15 principal components (PC) of the OD accounting for 90% of the original variability. The performance of the three ML models was measured with the area under the curve and continuous Boyce index. We collected 663 nonduplicated presence locations of Eastern wild turkeys ( HabitatEcological niche modeling (ENM), including habitat suitability modeling, has become a fundamental tool for understanding the spatial distribution and conservation of biodiversity. Habitat suitability models (HSMs) relate species occurrences to landscape variables or resource availability in space  to each of the random samples, and then aggregates the “votes” or averages results over all the trees to make classifications or numeric predictions Figure . Random Habitat suitability mapping often uses a large number of landscape variables  to predict habitat suitability. Many of those landscape variables are highly correlated to one another, leading to multicollinearity in habitat and resource selection models . The second method is to use the scores of orthogonal principal components, which explain the majority of variation in the original environmental variables . Drake et al.  is the largest galliform in North America , such as the entire state of Mississippi , has been reported in the literature. In this study, we first developed statewide habitat suitability maps with a large sample size of presence data  using MaxEnt, RFs, and SVMs. Second, we compared predictive performances of MaxEnt, RFs, and SVMs between correlation removal and principal component approaches to multicollinearity. Ecological studies have not exploited extensively the excellent performances of SVMs in pattern identification and recognition and the capacity to analyze large amounts of data and complex relationships , pine forests, and pine‐hardwood mixed forests. The Mississippi Alluvial Valley region in westcentral Mississippi was dominated by agriculture, with only ca. 19% of land being covered by remnant bottomland hardwood forests . Mississippi has a flat topology with elevation ranging from 0 to 2.2n = 17); (b) male bird harvest locations in March and April of 2014 (n = 74) and 2015 (n = 91); (c) brood surveys of females and young of the year birds in June, July, and August of 2014 (n = 288) and 2015 (n = 202); and (d) random sightings across the state throughout the year (n = 91). Cooperative turkey hunters recorded the geographic coordinates (longitude and latitude) of harvest locations on data sheets, which were designed and distributed by the senior author before the turkey hunting seasons (from mid‐March to 01 May), using a hand‐held global positioning system (GPS) unit. Impromptu sightings occurred when wildlife biologists of the Mississippi Department of Wildlife, Fisheries, and Parks (MDWFP) conducted routine work. Geographic coordinates of other sighting locations were determined using high‐resolution (15 m) Google Earth© Map (http://www.earth.google.com). Brood surveys were conducted by the MDWFP wildlife biologists in June, July, and August. Wild turkey broods with females were detected ~100–150 m from observers. Geographic coordinates of detected broods were recorded using a hand‐held GPS unit. Location errors  were less than the 250‐m resolution of the land cover and land use (LCLU) maps used in our study. Additionally, frequency, edge density, and distance of land covers were generated as averages within a 1,785‐m circular buffer, which is the radius of average annual home range of wild turkeys in Mississippi  wild turkey trapping locations in January, February, and March of 2009 and 2010 . Mississippi LCLU types included 15 classes: open water, developed open space, developed low intensity, developed medium intensity, developed high intensity, barren land, hardwood forest, pine forest, mixed forest, shrub/scrub, grassland/herbaceous, pasture/hay, cultivated crops, woody wetland, and emergent herbaceous wetlands  satellite imagery classified by the Multi‐Resolution Land Characteristics Consortium  habitat suitability modeling compared to other spatial scales.We generated 250‐m LCLU raster maps (or layers) by resampling from the original 30‐m LCLUs to reduce computational burdens. We then derived three landscape variables for each of the nine LCLU classes: distance to the nearest grid cell (m), relative frequency (0–1.0), and edge density (m/ha), producing a total of 27 landscape variables . Distance layers were generated using the program Biomapper module DistAn  to transform the original 27 landscape variables to principal components (hereafter PC data), which were orthogonal to one another, to avoid multicollinearity among original landscape variables. PC data were generated using the geographic information system (GIS) software IDRISI 15.0 , which generates the raster images of PCs in the same file format as programs CircAn and DistAn.uSDM to calculate VIFs of 27 landscape variables  to remove landscape variables which were highly correlated with other landscape variables, decreasing the extend of multicollinearity  uses environmental conditions including landscape variables at presence locations to quantify the multi‐dimensional ecological characteristics of the occupied habitat  to 10,000. We used the default value of the parameter mtry . At last, RFs aggregate the results over 10,000 trees to make predictions, taking the majority of the votes of 10,000 trees for classification , respectively, with 453 presence locations coded as 1's) and 453 selected pseudo‐absence locations (coded as 0's) using the R package 's and 45svm of the R package e1701 , using the function 2.5We evaluated the predictive accuracy of ENFA, RF, MaxEnt, and SVM predictions using the same test data (210 nonduplicated presence locations) with the continuous Boyce index  index from receiver operating curve (ROC) to assess the accuracy of ENFA, MaxEnt, RFs, and SVMs Hilden, . The ROCevaluate. We generated Boolean maps of suitable habitat, having the value 1 or 0 for a grid cell with its suitability index being greater or less than the threshold.We also determined the HSI threshold by maximizing the sum of the true positive rate and true false negative rates of each habitat suitability model using the function 3The first 15 principal components (PCs) explained 90% of variability in the original 27 landscape variables. The variable inflation factors (VIF) of 14 original landscape variables were greater than the cutoff of three and were excluded from the reduced data  with RFs slightly over performing MaxEnt and SVMs , random forests (RFs), and support vector machines (SVMs) for habitat suitability modeling. Neither multicollinearity nor correlation removal reduced the predictive performance of MaxEnt, RFs, and SVMs substantially. Additionally, partial‐dependent effects of distance to hardwood forest and hardwood forest edge density are consistent between the RF models using the original data with multicollinearity and the reduced data of independent predictors. The occurrence of wild turkeys exhibited an increase and then level‐off with increasing hardwood proportion and edge density . Low amounts of hardwood forest and edge density appeared to limit the habitat use of wild turkeys. Nevertheless, the benefits of increasing hardwood forests and edge density leveled off or became saturated at high levels, consistent with the prediction of the functional response hypothesis for animal habitat selection  has various algorithms to combat the curse of dimensionality and multicollinearity including regularization and bagging. MaxEnt developed by Phillips et al.  used thed Hoarau . Francisd Hoarau  and thisRandom forests may outperform SVMs and MaxEnt in ecological classification primarily because of the bagging algorithm Breiman, , although, a reduced dimensionality of input data, to achieve sparsity. This study demonstrated robust predictive performance of SVMs to landscape data of collinearity like Drake et al. . In our study, hardwood forest edge density served as a surrogate for the relative simultaneous access to both hardwood forests and different land covers that wild turkeys may have found within their home ranges. Landscapes of <20% or >30% hardwood forests may lack diversity, which reduced hardwood edge density, and thereby negatively affected the occurrence probability and potential abundance of wild turkey.Occurrence probabilities of wild turkey were also limited by low hardwood forest edge density below about 30 m edge/ha Figure . Davis ePinus sp.) or hardwood forests (Miller & Conner, The abundance–suitability relationship may be positive in wildlife, including birds and mammals (Weber et al., The authors have no conflict of interest related to this work.GW and AF conceived the ideas. AF, GW, and DG designed the study. AF collected data. DG and AB coordinated and participated in statewide data collection. AF and GW analyzed data. AF drafted the manuscript. All authors contributed to writing, revising, and improving the manuscript and gave the final approval for publication. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


These results indicate that infections caused by multidrug resistant microorganisms could be treated using a synergistic combination of antimicrobial drugs and AgNPs. Further studies are necessary to evaluate the specific mechanisms of action, which could help predict undesirable off-target interactions, suggest ways of regulating a drug’s activity, and identify novel therapeutic agents in this health problem.The increase in the prevalence of bacterial resistance to antibiotics has become one of the main health problems worldwide, thus threatening the era of antibiotics most frequently used in the treatment of infections. The need to develop new therapeutic strategies against multidrug resistant microorganisms, such as the combination of selected antimicrobials, can be considered as a suitable alternative. The in vitro activities of two groups of conventional antimicrobial agents alone and in combination with silver nanoparticles (AgNPs) were investigated against a set of ten multidrug resistant clinical isolate and two references strains by MIC assays and checkerboard testing, as well as their cytotoxicity, which was evaluated on human fibroblasts by MTT assay at the same concentration of the antimicrobial agents alone and in combination. Interesting results were achieved when the AgNPs and their combinations were characterized by Dynamic Light Scattering (DLS), Zeta Potential, Transmission Electron Microscopy (TEM), UV–visible spectroscopy and Fourier Transforms Infrared (FTIR) spectroscopy. The in vitro activities of ampicillin, in combination with AgNPs, against the 12 microorganisms showed one Synergy, seven Partial Synergy and four Additive effects, while the results with amikacin and AgNPs showed three Synergy, eight Partial Synergy and one Additive effects. The cytotoxic effect at these concentrations presented a statistically significant decrease of their cytotoxicity ( Urinary tract infections (UTIs) are defined as inflammatory processes related to the invasion and multiplication of microorganisms that occur at any level of the urinary tract, including urethral (urethritis), bladder (cystitis), ureters (ureteritis) and kidney infections (pyelonephritis) .Approximately between 150 and 250 million cases of UTIs occur each year worldwide . RecentlEscherichia coli (UPEC) is the major microorganism isolate, which is a member of the Enterobacteriacea family [Klebsiella sp. and Proteus mirabilis, both of which are characterized by their urease enzyme and Gram-positive bacteria such as Staphylococcus saprophyticus and Enterococcus faecalis [Gram-negative intestinal bacteria are the most common etiology agents of UTIs, where uropathogenic a family . Other cfaecalis .Escherichia coli, Pseudomonas aeruginosa, Enterococcus, Proteus mirabilis, Klebsiella pneumoniae and coagulase-negative staphylococci [Urinary Tract Infections Associated with Catheters (CAUTI) are one of the most frequent explanations of nosocomial infections . Patientylococci ,16.The antibiotic resistance of bacteria is a global health problem that is continually expanding, and is recognized as a medical problem that increases morbidity and mortality rates, which implies length of hospital stays as well as cost and bad prognosis ,18. In fIn this context, nanotechnology opens new possibilities, allowing new solutions with old resources. Nanoscale materials such as silver nanoparticles (AgNPs) have emerged as novel agents due to their unique physicochemical properties and remarkable antimicrobial activities that confer a great advantage for the development of alternative products against, for example, multi-drug resistant microorganisms ,20. Due Recently, several studies have indicated that AgNPs can enhance the effect of antibiotics against susceptible and resistant bacteria,  as a welSome authors have reported that AgNPs have toxic effects on mammalian cells; for example, impairment of normal mitochondrial function, increased membrane permeability and generation of reactive oxygen species ,25.In this study, the synergistic activity of AgNPs was evaluated with conventional antibiotics against Gram-positive and Gram-negative multi-drug resistant isolates from clinical samples. Results presented here show that AgNPs, in combination with antibiotics, increase the antimicrobial effect in an additive or synergistic manner. Furthermore, MTT assays suggest that at low concentrations the AgNPs and their combinations do not present cytotoxic effects in eukaryotic cells. TEM micrograph revealed that the AgNPs were of spherical and pseudospherical shapes . Based oThe particle size distributions of AgNPs + AMP and AgNPs + AMK were also evaluated in Dulbecco’s Modified Eagle Medium (DMEM).AgNPs synthesized in aqueous solution and their combinations with antibiotics were characterized by DLS . The resThe hydrodynamic diameters and zeta potentials of AgNPs and their combinations were also evaluated in DMEM.The UV–visible spectrum revealed a peak at a wavelength of 412 nm for AgNPs whereas in the combination AgNPs + AMP, the peak was observed at 410 nm and in the combination AgNPs + AMK, the peak appeared at a wavelength of 450 nm . These pIn this study, gallic acid was used as a reducing and stabilizing agent, a molecule with a carboxylic group and rich in hydroxyl functional groups. Yoosaf et al. proposed that AgNPs are stabilized by gallic acid through electrostatic interactions through their oxidized carboxylic group and the afore-cited hydroxyl groups were capable of forming hydrogen bonds . −1 disappeared completely  showed a MIC to AgNPs between 4 and 16 μg/mL. The bacterial strains showed MIC values of 4–128 μg/mL for amikacin and all Gram-negative strains were resistant to ampicillin. A set of ten clinical pathogenic strains resistant to antibiotics associated with CAUTI were evaluated, of which two corresponded to Gram-positive strains and the rest to Gram-negative strains. The results showed that all clinical isolates  and E. coli (ATCC 25922). Synergistic interactions of AgNPs and ampicillin were found only against Acinetobacter baumannii. Other combinational activities of AgNPs and antibiotics were considered as partially synergistic interactions. These synergistic activities of AgNPs in combination with conventional antibiotics suggest that it may be possible to reduce the viability of bacterial strains at lower antibiotic concentrations  index . Synergitrations .p < 0.05). However, combinations of 128 μgAgNPs/mL + 64 μg of antibiotic/mL and 64 μgAgNPs/mL + 32 μg of antibiotic/mL caused a statistically significant decrease in cell viability when compared with the rest of the combinations tested (p < 0.05) evidenced by the reduction of the mitochondrial activity. On the other hand, it is important to highlight that when AgNPs were combined with antibiotics, at concentrations equal to or less than 8 μg AgNPs/mL showed a viability percentage between 90–95%  were tested at concentrations of 64, 32, 8, 2, 0.5 and 0.125 μg/mL in human fibroblasts. It was found that the viability percentage for each of the concentrations of ampicillin was greater than 80% and for amikacin greater than 76%. To evaluate the cytotoxic effects of the combination of AgNPs and conventional antibiotics, ten combinations of different concentrations  were tested. It was found that there is no statistically significant difference between the two treatments (AgNPs + AMP and AgNPs + AMK) with respect to cell viability when the two-way ANOVA was performed  was used as a metallic precursor and gallic acid (0.1 g) was used as a reducer and stabilizer agent. NaOH (3 M) was used for pH regulation. AgNPs were synthesized by dissolving 0.0169 g of AgNO3 in 90 mL of deionized water and this solution was placed in a 250 mL reaction vessel. A total of 0.01 g gallic acid was dissolved in 10 mL of deionized water and was added to the AgNO3 solution with magnetic stirring. After the addition of gallic acid, the pH value of the solution was adjusted using a solution of NaOH 3 M. At the end of the synthesis, approximately 100 mL of nanoparticles were obtained with a pH of 12.66, of which 50 mL were dialyzed for 48 h on a 12 kDa nitrocellulose membrane.For the synthesis of nanoparticles, AgNO−1 with a resolution of 2 cm−1 and 200 times scanning using the attenuated total reflection (ATR) method.AgNPs were characterized by Dynamic Light Scattering (DLS), the hydrodynamic diameter and zeta potential were determined using a Malvern Zetasizer Nano ZS  operating with a He-Ne laser at a wavelength of 633 nm and a detection angle of 90°. Samples were analyzed for 60 s at 25 °C. To confirm the shape, each sample was diluted with deionized water and 50 μL of each suspension was placed on a copper wire for Transmission Electron Microscopy (TEM). All samples were analyzed by Transmission Electron Microscopy  at an acceleration voltage of 100 kV. Afterwards, AgNPs were characterized by UV-visible spectroscopy using an S2000 UV-Vis spectrophotometer from OceanOptics Inc. . The functional groups present in the antibiotics were identified by Fourier Transform Infrared Spectroscopy . A certain amount of nanopowder was collocated in the equipment and the spectrum was taken in the range of 400–4000 cmTo study the effect of ampicillin and amikacin on the size, shape and stability of the AgNPs, an aqueous solution containing a 1:1 ratio of antibiotic (128 μg/mL) and nanoparticles (128 μg/mL) was prepared for each antibiotic. These solutions were characterized by TEM, DLS, zeta potential and UV-visible spectroscopy. On the other hand, the chemical interaction between the AgNPs and antibiotics was carried out by FTIR, we prepared an aqueous solution containing higher concentrations of antimicrobials (500 μg/mL), the combinations preserved the ratio of 1:1. Subsequently, these solutions were centrifuged, keeping only the precipitate, which was left to dry for 24 h at room temperature.The study protocol and the letter of informed consent were approved by the local Hospital’s Ethics and Science Committee with the number 102-16.The study included urine samples from patients with urinary catheters treated at the local Hospital. Patients were selected in basis to NOM-045-SSA2-2005. For the purposes of this study, only samples of patients older than 15 years, of any genus, whose culture had a bacterial count greater than or equal to 50,000 CFU/mL were selected, and also with an antimicrobial resistance profile.Urine samples were collected in 5 mL syringes using the probe puncture technique under aseptic conditions by trained personnel. Microbiological analysis was performed according to the established criteria by the American Society for Microbiology (ASM) for urine samples. A count greater than or equal to 50 colonies  was considered as the cutoff point for diagnosing infection. Plaques without development at 24-h incubation were discarded and indicated absence of urinary tract infection.The identification and antimicrobial susceptibility of the microorganisms isolated were determined by VITEK2 equipment. From a pure culture grown for 24 h, an inoculum was transferred to a test tube with 3 mL of solution sterile saline . Subsequently, the turbidity was adjusted to 0.50–0.63 units of the McFarland scale with the densitometer. The bacterial suspension was placed inside the cassette. The identification and susceptibility cards were placed in the nearby slot, inserting the transfer tube into the test tube with the corresponding suspension. The cassette was placed with the samples in the VITEK2 system. The resistance profiles are shown in The conservation of the strains was carried out by the freeze conservation method. Glycerol was used as the cryoprotective agent. The samples were stored at −20 °C.Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922. The antibiotics used were oxacillin (64 μg/mL) and ceftazidime (32 μg/mL). The assay was performed in duplicate for four days. The results of the standardization of the MIC are shown in The Minimum Inhibitory Concentration (MIC) was determined by broth microdilution assay in accordance with the procedures recommended by the Clinical and Laboratory Standards Institute (CLSI) . MICs we5 CFU/mL) in 100 μL Mueller–Hinton broth and incubated at the optimum temperature and time of growth conditions (37 °C/24 h). The MIC was determined as the least dilution without any turbidity. The MICs of single antimicrobial A and B (MICA and MICB) and in combination were determined. The ten multidrug resistant clinical strains were exposed to serial dilutions of the antimicrobial agents. Subsequently, we calculated the proportion: MICAntibiotic alone/MICAntibiotic in combination (fold-change) to describe the number of times that MIC decreased from an initial to final value.The MIC of each antimicrobial substance alone or in combination was determined by a broth microdilution method in accordance with CLSI standards. The assay was performed in 96-well microtiter plates, a two-fold dilution of the antibiotic was distributed into each well to obtain a varying concentration of 128, 64, 32, 16, 8, 4, 2, 1, 05, 0.25 and 0.125 μg/mL in the wells of the first row, while those of the AgNPs were similarly distributed among the first column (128 to 1 μg/mL). The AgNPs dilutions were started from the columns to the right and the antibiotic dilutions were started from the first row downwards. Thus, each of the wells held a unique combination of concentrations of AgNPs and the antibiotic. The broth microdilution plates were inoculated with each test microorganism to yield the appropriate density  were used. Cells were dispensed in 96-well microplates at a density of 5000 cells per well in DMEM supplemented with 1% fetal bovine serum (FBS) for 24 h at 37 °C. After 24 h the cells were incubated with the established concentrations of AgNPs and antibiotics for 24 h at 37 °C. Treatment was withdrawn and 100 μL MTT -2,5-diphenyl tetrazolium bromide) (0.5 mg/mL) was added in DMEM without FBS for 4 h at 37 °C for the formation of formazan crystals. MTT was removed from the wells and 100 μL of DMSO was added to read absorbance in a Synergy H1 microplate reader with Gen 5 software  at a wavelength of 595 nm. As a positive control, cells were treated with hydrogen peroxide and as a negative control, they were only treated with medium. The assay was performed in triplicate. p < 0.05 was considered significant. Each assay was repeated three times. Data are presented as mean ± standard deviation (SD). The comparison between the effects of the two sources of variation was made using the two-way analysis of variance (ANOVA). The analysis was performed with the statistical software SPSS 23.0 . A value of These results indicate that infections due to multidrug resistant microorganisms could be treated by the use of a synergistic combination of antimicrobial drugs and AgNPs. Further studies are necessary to evaluate the specific mechanisms of action, which could help predict undesirable off-target interactions, suggest ways of regulating a drug’s activity, and identify novel therapeutics to this health problem.


To assess the frequency of choledocolithiasis and the role of preoperative laboratory findings, ultrasound (US) and magnetic resonance cholangio-pancreatography (MRCP) in the detection of choledocolithiasis in patients with gallbladder stones awaiting cholecystectomy.2) test was performed to assess the statistical significance of differences in the frequency of choledocolithiasis based on laboratory findings, choledocal diameter on US and group risk.A consecutive sample of 104 patients underwent MRCP prior to cholecystectomy. The patients were classified into different groups on the basis of the risk of choledocolithiasis. A specialised doctor with more 10 years of experience performed the US interpretation and a radiologist performed the MRCP interpretation blinded to US or aspartate aminotransferase (AST)/alanine aminotransferase (ALT)/alkaline phosphatase (ALP) results. A chi-square , with no statistically significant differences between the high/moderate risk and low/no risk groups and between the patients with normal and altered laboratory findings or choledocal diameter on preoperative US. The sensitivity and specificity of AST/ALT , ALP , total serum bilirubin  and choledocal diameter  were, respectively, 28.6 and 94.8%, 85.7 and 17.5%, 14.3 and 93.8%, and 14.3 and 95.9%.MRCP is a reliable evaluation for the detection of common bile duct (CBD) stones, reducing the misdiagnosis of retained choledocholithiasis with normal biochemical predictors and US examination.• MRCP is a non-invasive method for the detection of CBD stones.• Preoperative MRCP reduces the misdiagnosis of retained choledocholithiasis.• Detection of choledocholithiasis is mandatory prior to cholecystectomy to avoid surgical morbidity Common bile duct (CBD) stones may occur in up to 5–15% of patients with symptomatic gallstone disease .Magnetic resonance cholangio-pancreatography (MRCP) is a reliable tool to study the biliary tree, with a diagnostic accuracy of almost 100% in demonstrating CBD stones , and preWe collected preoperative laboratory findings and investigated routinely our patients through MRCP and US prior to cholecystectomy, in order to answer the following questions: what is the predictive value of liver function tests and morphological features on abdominal US for CBD stones? What is the frequency of CBD stones? Is it possible to identify patients at risk of choledocholithiasis? Is it useful to perform preoperative MRCP in all patients before cholecystectomy?We prospectively collected the data of patients who underwent cholecystectomy because of a gallstone disease between January 2012 and December 2013 at the “Regina Pacis” Clinic, San Cataldo (CL), Italy. Data collected included clinical characteristics, preoperative serum total bilirubin, alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and choledocal diameter on preoperative US.Among 149 patients, 45 could not undergo MRCP because of claustrophobia, certain pacemakers, implantable cardiac defibrillators or deployable metallic prostheses. Thus, only 104 patients  were included in the study and underwent preoperative unenhanced MRCP .If a CBD lithiasis was found, preoperative extraction by endoscopic retrograde cholangio-pancreatography (ERCP) was attempted; then, laparoscopic cholecystectomies were performed by two surgeons.The details of all patients were entered into a spreadsheet (Excel).We divided the patients into different risk groups as suggested by Kim et al. in 2002 : high, mThe study was approved by the institutional ethics committee.The statistical analysis was performed by using IBM SPSS Statistics 20.0.2) test was performed to assess the statistical significance of differences in the frequency of CBD lithiasis based on laboratory findings, choledocal diameter on US and group risk according to Kim et al.’s classification. A p-value < 0.05 (χ2 > 3.84) was considered significant. Moreover, we assessed the positive and negative predictive values  of biochemical abnormalities and morphological features on abdominal US.A chi-square , but none of them demonstrated cholangitis.Regarding the preoperative laboratory findings, 17 patients had elevated AST (range 42–275 IU/L) and ALT (range 51–382 IU/L), 86 patients had elevated ALP (range 112–900 IU/L) and 18 patients had elevated total serum bilirubin (range 1.3–6.6 mg/dL).On preoperative US examination, 5 out of 104 patients demonstrated a dilated CBD and, among them, only one patient 20%) presented CBD lithiasis on MRCP, whereas 6 out of 99 patients (6%) with normal choledocal diameter showed CBD lithiasis , ALP , total serum bilirubin  and choledocal diameter on preoperative US .With respect to the frequency of CBD lithiasis, MRCP showed calculi in 7 out of 104 patients with gallbladder stones awaiting cholecystectomy (6.7%), with no statistically significant differences between the groups with normal and altered AST/ALT , ALP , total serum bilirubin  and choledocal diameter  were, respectively, 28.6 and 94.8%, 85.7 and 17.5%, 14.3 and 93.8%, and 14.3 and 95.9% . The frequencies of CBD lithiasis in each group are presented in Table So the patients were classified into four different risk groups and the difference in frequency of CBD lithiasis was not statistically significant between the high/moderate risk and low/no risk groups .Greater accuracy can be obtained by using laboratory findings in addition to imaging modalities , 17.Non-invasive imaging techniques such as US are widely used for the diagnosis and monitoring of many biliary diseases. A CBD diameter greater than 6 mm on US is associated with a higher prevalence of choledocholithiasis .However, these techniques have limitations, such as the low sensitivity of US for detecting CBD calculi, detecting only from 33 to 55% of the CBD stones . In our Computed tomography is associated with ionising radiation and is unreliable for detecting non-calcified stones.ERCP was accepted as the ‘gold standard’ for bile duct imaging, with the advantage of permitting the bile duct to be cleared of stones. However, it is an invasive technique, with a reported mortality rate of 0.1–3%  and compMRCP is a non-invasive imaging technique that does not require the use of X-rays or contrast media. The most important drawbacks of MRCP are its inability to offer therapeutic interventions and its high cost.The criteria for a positive MRCP were signal defects within the CBD, defined variably as foci or rounded and oval in some studies .MRCP has been shown to demonstrate normal and variant biliary anatomy accurately, as well as benign and malignant causes of bile duct obstruction with a sensitivity and specificity of 95–100% in the detection of CBD stones , 26.In 2003, Romagnuolo et al.  reportedAccuracy in detecting CBD stones, absence of ionising radiation and greater safety make MRCP a competitive diagnostic method in patients with biliary obstruction.To our knowledge, there are different studies evaluating prospectively a scoring system designed to improve the accuracy of CBD stone prediction before laparoscopic cholecystectomy and to minimise the number of preoperative MRCP , 29.However, we tried to use our data and insert them into a scoring system , showingThis strong concealment may lead to serious consequences for patients and associated overall healthcare costs .This article does not analyse the cost-effectiveness for patients; however, based on this study, the importance of preoperative MRCP as the only non-invasive tool in screening patients with suspected choledocholithiasis was clearly demonstrated. MRCP can allow the surgeon to know the state of the patient’s biliary ductal condition and also the possible presence of severe inflammation that is reported as one of the most important reasons for bile duct injury .For this reason, MRCP routine use, besides being diagnostically useful in the perioperative management in some cases, might be justified, despite its cost.MRCP is a reliable and non-invasive evaluation for the detection or exclusion of CBD stones, reducing the misdiagnosis of retained choledocholithiasis with normal biochemical predictors and normal US examination. Thus, routine preoperative MRCP examination is suggested in patients who are candidates for cholecystectomy because of a gallstone disease.