To assess whether strain histograms are equal to strain ratios in predicting breast tumour malignancy and to see if either could be used to upgrade Breast Imaging Reporting and Data System (BI-RADS) 3 tumours for immediate biopsy.Ninety-nine breast tumours were examined using B-mode BI-RADS scorings and strain elastography. Strain histograms and ratios were assessed, and areas- under-the-receiver-operating-characteristic-curve (AUROC) for each method calculated. In BI-RADS 3 tumours cut-offs for strain histogram and ratio values were calculated to see if some tumours could be upgraded for immediate biopsy. Linear regression was performed to evaluate the effect of tumour depth and size, and breast density on strain elastography.Forty-four of 99 (44.4%) tumours were malignant. AUROC of BI-RADS, strain histograms and strain ratios were 0.949, 0.830 and 0.794 respectively. There was no significant difference between AUROCs of strain histograms and strain ratios (P = 0.405), while they were both inferior to BI-RADS scoring . Four out of 26 BI-RADS 3 tumours were malignant. When cut-offs of 189 for strain histograms and 1.44 for strain ratios were used to upgrade BI-RADS 3 tumours, AUROCS were 0.961 (Strain histograms and BI-RADS) and 0.941 (Strain ratios and BI-RADS). None of them was significantly different from BI-RADS scoring alone (P = 0.249 and P = 0.414). Tumour size and depth, and breast density influenced neither strain histograms  nor strain ratios Strain histogram analyses are reliable and easy to do in breast cancer diagnosis and perform comparably to strain ratio analyses. No significant difference in AUROCs between BI-RADS scoring and elastography combined with BI-RADS scoring was found in this study. Elastography has emerged as an auspicious adjunct to B-mode ultrasound examination –3. UltraTumour stiffness is correlated with the risk of malignancy , 2, 5. BIn this study we hypothesized that the diagnostic accuracy of strain histograms was comparable to that of strain ratio calculations. We also evaluated if tumour size, tumour depth and breast density had any impact on the elastographic evaluations. The ultimate goal of the study was to assess whether strain elastography could be used to upgrade stiff BI-RADS 3 tumours to immediate biopsy without lowering the diagnostic accuracy.This study was approved by the Regional Ethical Committee on Medical Research (reference id: H-2-2013-FSP46). Patients gave informed oral consent before participation. Written consent was waived by the regional ethics commitee, because strain elastography was part of the examination set-up prior to the initiation of the study. Oral consent was documented in the patients’ medical records. This procedure was approved by the regional ethics committee.Between August 2013 and March 2015 99 women ) suspected of breast cancer and scheduled for biopsy with 108 breast tumours were prospectively and consecutively included. To avoid a clustering effect, only one tumour from each woman was randomly included for further analysis. This yielded 99 tumours for final evaluation. Women older than thirty years of age had mammography performed prior to the ultrasound examination. The same radiologist (SS > 15 year’s experience in breast radiology) performed all mammography-, ultrasound- and elastography examinations including all biopsies. Biopsies were performed as fine needle aspiration (14 tumours) or histological biopsy or excision (85 tumours) according to national guidelines. Triple consensus of diagnostic imaging (ultrasound and mammography), palpation and cytology was used as gold standard for all . Tu. Tu16]. All BI-RADS 3 tumours were evaluated to see if strain histograms and ratios could potentially be used to upgrade stiff tumours to immediate biopsy. For both strain histograms and strain ratios a cut-off just below the lowest value of the softest malignant tumour was found, and all tumours above this cut-off were reclassified as BI-RADS 4. All tumours below the cut-off remained BI-RADS 3. All other BI-RADS scorings were left unchanged.th edition [Most patients had mammography performed on the same day, prior to the ultrasound examination. For patients who did not have mammography performed on the same day as the biopsy, mammograms up to six months old were used for breast density evaluation. Breast density was assessed by visual inspection according to the American College of Radiology BI-RADS Atlas 4 edition .Descriptive statistics were calculated using Microsoft Excel version 2011 . The statistical software R version 0.98.1091  was used for all other statistical analyses. Receiver-operator-characteristics (ROC curves) were done in SPSS version 20 . In case of skewness in the distribution of data, data were log-transformed. Averages are presented as median and/or mean (+/- SD) and a range if relevant. Differences between means were assessed using an unpaired t-test. For evaluation of diagnostic performance, area-under-receiver-operator-characteristics (AUROC) curves were performed. For BI-RADS scoring sensitivity, specificity, positive predictive value and negative predictive value was calculated using a cut-off between 3 and 4. To assess the impact of tumour depth and tumour diameter on strain ratios and strain histograms, linear regression with backwards elimination of insignificant covariates was performed. To assess the impact of breast density, analysis of variance was performed. The significance level was set at 0.05.Fifty-five of ninety-nine tumours were benign ) and 44 were malignant ). Histological diagnoses are summarized in There was a significant difference between means of benign and malignant tumours for strain histograms and strain ratios (P < 0.001 for both) . For B-mFour of 26 tumours classified on ultrasound as BI-RADS 3 were malignant . By usinThis study evaluates the diagnostic performance of strain histograms, strain ratios and ultrasound BI-RADS scoring in a population of patients undergoing biopsy of a breast tumour. AUROCs showed that strain histograms and ratios were equal in discerning malignant from benign breast tumours, but were inferior to BI-RADS scoring.When using strain elastography to upgrade BI-RADS 3 tumours to BI-RADS 4 and immediate biopsy, strain histograms was were able to do so with fewer benign tumours being reclassified as BI-RADS 4. The combined AUROCs of BI-RADS scoring and strain histograms and BI-RADS scoring and strain ratios were not significantly different from that of BI-RADS scoring on its own. The study was not powered to assess the different ability to successfully reclassify BI-RADS 3 tumours for immediate biopsy between strain histograms and strain ratios.Tumour maximal diameter, depth and mammography breast density had no effect on strain histograms or ratios. Other studies have found improved diagnostic performance when combining elastography and BI-RADS-scoring , 17, 18.Few studies have investigated the application of strain histograms in breast cancer diagnosis –22. In mMeta-analyses have shown that both strain and shear-wave elastography perform well in breast cancer diagnosis , 23, 24.Strain ratios provide a semi-quantitative measure of tumour stiffness, which is desirable for malignancy risk stratifications , 8. RepoWhen combining a new diagnostic test with an established one, such as the BI-RADS classification, a key matter to consider is how to combine the two tests. Studies have used reclassifications of either BI-RADS 3 or the BI-RADS 4a subclass , or both, according to elastography assessments, as these classes have the most false negative and false positive results , 17, 18.For strain ratio calculation, the two ROIs are subject to equal scanning conditions and are therefore theoretically unaffected by anything but the difference in tissue strain. As strain histograms are calculated from a single ROI, tumour depth and size, and breast density could have an impact on strain histogram values. In this study neither tumour depth or size, nor breast density had an impact on strain histograms and strain ratios. In agreement with this, phantom studies have shown, that lesion size carries little impact in strain histogram and strain ratio values , 29. AltFour out of 26 BI-RADS 3 tumours were malignant which is a high fraction of malignant BI-RADS 3 tumours compared to other studies, which may be due to a small sample size. Larger studies are needed to further clarify the reason for this high malignancy rate. The sensitivity and AUROC for BI-RADS scoring found in this study is comparable to the ones reported in previous work, and specificity was relatively high, signifying that the overall BI-RADS classification was performed adequately . A largeMany studies of ultrasound elastography in breast tumour diagnosis use only histological biopsy as gold standard. In the Scandinavian countries there has been a long tradition of using triple testing in breast diagnosis consisting of palpation, imaging (mammography and/or ultrasound) and fine-needle aspiration in suspicious tumours. Studies have shown that triple testing is equally certain to histological biopsy. We chose therefore to include tumours on which triple testing was used as gold standard.Strain histogram analysis was performed off-line in this study, which makes the analysis time-consuming and unfit for clinical use. Strain histogram software is however available for some ultrasound systems and should be easy to implement in a clinical set-up.The study was performed in a specialized breast radiology unit, with a high frequency of malignancies. A strength of the study was that only one, highly experienced breast radiologist performed all examinations, but the results could differ for less experienced operators in less specialized departments. It is a limitation of this study that no inter- or intraobserver evaluation of strain histogram analysis was undertaken. Previous studies have found that both intra- and interobserver agreement is an issue with both semi-quantitative and qualitative strain elastography while shear-wave elastography generally shows less variability –33. To rStrain histogram analyses are reliable and easy to do in breast cancer diagnosis and perform comparably to strain ratio analyses. Strain histograms and ratios are unaffected by tumour size and depth, and breast density assessed by mammography. No significant difference in AUROCs between BI-RADS scoring and elastography combined with BI-RADS scoring was found in this study.


Despite significant changes in the therapeutic landscape of renal cell carcinoma, the majority of patients with metastatic disease eventually progress after first-line treatment with vascular endothelial growth factor receptors (VEGFR) tyrosine kinase inhibitor (TKI) therapy. Understanding existing data on subsequent therapies is crucial to define an optimal treatment sequence following first-line failure. This review examines the data supporting currently approved agents in this setting and provides a framework for decision-making regarding treatment sequencing beyond first-line therapy with VEGFR TKIs. Significant changes in the therapeutic landscape of renal cell carcinoma (RCC) have drastically improved the outcome of patients with advanced RCC. Since the approval of high-dose IL2 (HD IL-2) in the mid-1990s . These iThe endothelial growth factor receptors (VEGFR) tyrosine kinase inhibitors (TKIs) sunitinib and pazopanib are approved for the vast majority of patients with untreated advanced RCC and are the standard of care in this setting , 7. TemsIn the setting of disease progression on VEGF-directed therapy, multi-kinase inhibition has become an attractive therapeutic option with agents such as cabozantinib, a VEGFR, MET, and AXL inhibitor . SimilarEverolimus is an oral mTOR inhibitor, a key molecule that mediates cellular growth, proliferation, cellular metabolism, and angiogenesis . It exerPFS assessed by independent central review was significantly longer for those receiving everolimus compared to those on placebo . The PFS benefit was observed and maintained across pre-defined sensitivity and subgroup analyses. The Response Evaluation Criteria In Solid Tumors (RECIST)-defined  ORR was The phase III AXIS study evaluated the clinical efficacy and safety of axitinib versus sorafenib as a second-line therapy . AxitiniRecently, Motzer et al.  reportedper day. Patients were stratified based on the standard disease features for advanced RCC studies. The primary endpoint was PFS with secondary endpoints that included ORR, OS, and safety. With a median follow-up of 9.2 months, the median PFS was 4.3 months for temsirolimus and 3.9 months for sorafenib . The confirmed independent review ORR was 8% and this was similar in both arms. The median OS was significantly longer with temsirolimus than with sorafenib with 12.3 versus 16.6 months, respectively . No new safety signals of concern were identified. In fact, the same proportion of patients experienced at least one G3 AE (70% with temsirolimus and 69% with sorafenib). G3 anemia and hyperglycemia were more common with temsirolimus . Similarly, G3 PPE was more frequent with sorafenib (0% vs. 15%). AEs resulting in dose reductions were observed in 16% of the patients receiving temsirolimus and 33% on sorafenib, respectively. Although the lack of correlation between PFS and OS in this trial is not well understood, INTORSECT was the first study in the second-line setting demonstrating an OS benefit for patients with advanced RCC. The results, however, do not suggest that sorafenib is the second-line agent of choice, rather solidified the hypothesis that angiogenic escape is in fact one of the mechanisms resistant to first-line VEGFR inhibition, and thus, the possibility that sequential VEGF-based therapy is a logical treatment strategy in patients with advanced RCC.Although temsirolimus has been approved as first-line treatment for untreated advanced RCC patients with multiple risk factors based on the OS benefit reported in the global AARC trial , its cliDovitinib is an oral TKI that inhibits FGF receptor (FGFR), VEGFR, and PDGFR. In preclinical studies, this agent demonstrated greater antitumor activity when compared to sunitinib and sorafenib , 24. DovIn the phase III GOLD trial, 284 patients with metastatic clear cell RCC who received one previous VEGF-targeted therapy and one previous mTOR inhibitor were randomized to receive either dovitinib 500 mg orally 5-days-on and 2-days-off or sorafenib 400 mg orally twice daily . PatientMost common G3/G4 AEs included hypertriglyceridemia (14%), fatigue (10%), HTN (8%), and diarrhea (7%) in the dovitinib group, and HTN (17%), fatigue (8%), dyspnea (7%), and PPE (6%) in the sorafenib group. Percentage of patients who discontinued dovitinib and sorafenib due to AEs is 15% and 12%, respectively. Despite the good tolerability, dovitinib failed to meet the primary endpoint of PFS versus sorafenib in patients pretreated and progressive RCC, and, consequently, has not been approved by FDA for the treatment of advanced RCC.Cabozantinib is an oral TKI of MET, VEGFRs, and AXL initially tested in a single–arm, phase I study of advanced RCC patients resistant to VEGF and mTOR inhibitors  that has>G3 AEs was similar for both drugs . Although HTN, diarrhea, and fatigue were the most common G3 AEs observed with cabozantinib, the most common G3 AEs reported with everolimus included anemia, fatigue, and hyperglycemia. Cabozantinib 60 mg per day eventually gained FDA approval in April 2016 and has become a standard of care in the second-line setting  and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) pathways in immune surveillance. This had led to the rapid development of agents capable of blocking these targets. A number of checkpoint inhibitors including ipilumumab (CTLA-4 inhibitor), atezolizumab (programmed death ligand 1 [PD-L1] inhibitor), and nivolumab and pembrolizumab (PD-1 inhibitors) have been approved for a variety of cancers. These antibodies block PD1 or its ligand PD-L1. Although atezolizumab and pembrolizumab are undergoing clinical development in RCC, nivolumab is the only PD1 inhibitor currently FDA approved in the VEGF TKI refractory RCC space. Nivolumab is a fully human IgG4 PD1 immune checkpoint inhibitor that selectively blocks PD1 expressed in T cells and PD-L1 and PD-L2 expressed in tumor cells and other immune cells .CheckMate-025, a phase III study, compared nivolumab to everolimus in patients with VEGF TKI refractory advanced RCC  9). A t. A t9). Nivolumab was also generally better tolerated than everolimus. G3/G4 treatment-related AEs occurred in 19% of the patients receiving nivolumab and in 37% of the patients receiving everolimus. The most common AE with nivolumab was fatigue (in 2%), whereas the most common AE with everolimus was anemia (in 8%). Furthermore, nivolumab was also associated with improved QOL compared with everolimus . Based oRecognizing the importance of VEGFR and mTOR pathways in RCC, a synergistic effect with dual inhibition has been suggested in preclinical and phase I and II studies . A recenPatients allocated to lenvatinib plus everolimus were treated for a median of 7.6 months, compared with 7.4 months for those assigned to single-agent lenvatinib and 4.1 months for those who received single-agent everolimus. Median PFS was 14.6 months for lenvatinib plus everolimus and 5.5 months for single-agent everolimus . Patients treated with single-agent lenvatinib had a median PFS of 7.4 months, which was also longer compared with those treated with single-agent everolimus . In an updated OS analysis, the median was 25.5 months for lenvatinib/everolimus, compared with 15.4 months for patients treated with everolimus , which demonstrated a trend towards survival benefit with the experimental combination  treatment-related AEs in patients allocated lenvatinib plus everolimus were diarrhea (20%), proteinuria (19%) in those assigned to single-agent lenvatinib, and anemia (12%) in those assigned to single-agent everolimus. Consequently, discontinuation rate secondary to AEs was 24% for patients treated with the combination regimen and single-agent lenvatinib, compared with 7% for patients treated with everolimus. Two deaths were possibly related to study drug, one cerebral hemorrhage in the lenvatinib plus everolimus group and one myocardial infarction with single-agent lenvatinib.Of note, there is no randomized phase III study confirming the results of this study. Therefore, limitations such as population selection and false-positive rates may had originated biased results , 42. NonMetastatic RCC (mRCC) has gained a variety of therapeutic options since the approval of the first VEGF TKI in the mid-2000s. Most recently, the treatment landscape for advanced RCC has drastically changed with results from three randomized clinical trials leading to the approval of nivolumab, cabozantinib, and the combination of lenvatinib/everolimus in previously treated advanced RCC . HoweverBecause RCC has joined the list of tumors where immune checkpoint inhibitors have shown to significantly improve OS and the possibility of long-term disease control for a subgroup of patients is real, the favorable side effect profile of these agents makes them a particularly appealing option. In opposition to other drugs, the majority of patients will develop minimal side effects; therefore, nivolumab may be considered for more fragile patients with lower performance status, multiple comorbidities, and organ dysfunction, including renal impairment, otherwise ineligible or borderline eligible for other therapies .Acknowledging that only a subgroup of patients potentially benefits from this class of agents and the absence of a reliable biomarker to help us selecting those patients, one of the questions that will need to be answered is the optimal timing of immunotherapy in RCC. Since its approval, most patients receive nivolumab as second-line agent with the expectation of an improved QOL and the possibility of a long-lasting response to therapy.Based on Checkmate 025, where patients had received up to three previous antiangiogenic therapies, approximately one-third of the patients were treated in third-line setting, suggesting a benefit later in the course of the disease. On the contrary, multiple clinical trials are ongoing, studying nivolumab and other checkpoint inhibitors in first-line setting, as single agents or in combination with VEGF inhibitors. We anticipate that the results of these studies may help answering this question of best timing for this class of agents , 46–48.>G3 AEs, whereas most patients receiving envatinib/everolimus (89%) reported significant AEs that led to dose reductions or interruptions. Similarly, nearly 20% had significant diarrhea, which led FDA to include a special warning on the drug’s label about the risk of this AE and mandated the investigation team to conduct a dose-finding study with an alternative dose combination  or SD with tumor burden reduction, regardless of performance status, number of prior lines, and metastatic sites  , 49, 50.bination . To overbination .Finally, the role of monotherapy with mTOR inhibitors has become less popular in advanced RCC and should be reserved for patients with molecular genotypes that would predict response to mTOR-based therapy (i.e. TSC1/2 mutations).Despite the array of approved drugs based on randomized trials, there are still no good data on sequencing strategies to support the use of a particular agent in the second and additional lines of therapy. Similarly, the multiple biomarker candidates from plasma, tumor, and host tissues have failed to identify patients more likely to respond to these therapies . PD-L1 aIn the absence of biomarkers that can predict response to existing agents, most physicians will define their second- and third-line therapy based on tolerability, AEs, QOL, and survival benefit. Based on randomized phase III data, nivolumab and cabozantinib are likely be the two agents of choice in these clinical scenarios. Nivolumab is a much more attractive option after TKI-based therapy when one looks at its AEs and compared those to the ones reported in the METEOR study. It is also mechanistically different to TKI-based therapy, and its broad utilization and activity across other solid tumors makes it a very attractive treatment alternative in the second-line setting after pazopanib- or sunitinib-based therapy. Others might feel that a rapid response is required especially for those patients who are primary refractory to first-line VEGFR TKI therapy. In this case, it is fair to say that cabozantinib can lead to rapid responses, often in less than 60 days. Main issues, however, remain tolerability as most patients do require dose reductions within the same time frame of 60 days. Although the misconception that checkpoint inhibitors cannot lead to rapid response remains, it is important to note that the median response to nivolumab in Checkmate 025 was 3.5 months. Similarly, best response at 4 months appears to predict for OS benefit as demonstrated in the subset analysis by Motzer et al. . As for With the introduction of different options in the VEGFR TKI refractory setting, choosing the ideal therapy remains a biologic and therapeutic challenge. There are currently no predictive biomarkers that determine the best therapy for the right patient, and little is known about the best sequence of treatments. Future clinical trials with drug combinations, predictive biomarkers, and novel therapeutics will hopefully help resolving some of these challenges.The authors declare no potential conflicts of interest with respect to research, authorship, and/or publication of this article.


To assess the feasibility of a streamlined strategy for improving tuberculosis (TB) diagnostic evaluation and treatment initiation among patients with presumed TB.Single-arm interventional pilot study at five primary care health centers of a streamlined, SIngle-saMPLE (SIMPLE) TB diagnostic evaluation strategy: 1) examination of two smear results from a single spot sputum specimen using light-emitting diode fluorescence microscopy, and 2) daily transportation of smear-negative sputum samples to Xpert® MTB/RIF testing sites.Of 1212 adults who underwent sputum testing for TB, 99.6% had two smears examined from the spot sputum specimen. Sputum was transported for Xpert testing within 1 clinic day for 83% (907/1091) of the smear-negative patients. Of 157 (13%) patients with bacteriologically positive TB, 116 (74%) were identified using sputum smear microscopy and 41 (26%) using Xpert testing of smear-negative samples. Anti-tuberculosis treatment was initiated in 142 (90%) patients with bacteriologically positive TB, with a median time to treatment of 1 day for smear-positive patients and 6 days for smear-negative, Xpert-positive patients.The SIMPLE TB strategy led to successful incorporation of Xpert testing and rapid treatment initiation in the majority of patients with bacteriologically confirmed TB in a resource-limited setting. PROMPT DIAGNOSIS AND TREATMENT of tuberculosis (TB) patients is essential for making progress towards TB elimination. However, 4.3 million of the estimated 10.4 million new cases in 2016 were not detected and reported to the World Health Organization (WHO).A principal reason for these failures with regard to care is the inadequacy of the current approach of using smear microscopy for TB diagnosis at primary health centers. First, smear microcopy has suboptimal sensitivity, identifying only about 50% of patients who actually have TB.12® MTB/RIF , a novel semi-automated molecular assay endorsed by the WHO in 2010.To address these limitations, there has been substantial donor investment in the scale-up of XpertThere is increasing evidence that behavioral theory and implementation frameworks are useful for identifying and targeting barriers to uptake of guidelines.In this pilot study, we introduced the SIMPLE TB strategy at five health centers in Uganda. Our objective was to evaluate the feasibility and implementation of each component.This single-arm interventional pilot study was conducted at five peripheral health centers (level IV) government health centers in rural Uganda. Level IV health centers are the lowest level of the health system where TB diagnosis (microscopy) and treatment are available, covering a population of approximately 100 000. These services are provided free-of-charge through the National Tuberculosis and Leprosy Programme (NTLP). Health centers were included in the pilot if they were 1) using standard (multi-day) sputum smear microscopy as the primary method of TB diagnosis; 2) participating in NTLP-sponsored external quality assurance (EQA) for sputum smear microscopy; and 3) affiliated with a district or regional hospital offering Xpert testing. We collected data on consecutive adults who were screened for chronic cough (>2 weeks) by the health center and who had sputum collected for TB diagnosis after the introduction of the two SIMPLE TB strategy components at each health center. Mycobacterial culture and speciation were not routinely performed, as they are not standard of care for TB diagnosis at the peripheral health centers.The study protocol was approved by the institutional review boards of the University of California San Francisco, San Francisco, CA, USA, and Makerere University, Kampala, and by the Uganda National Council for Science and Technology, Kampala, Uganda.boda boda) on a daily, rather than weekly, basis. Patients were instructed to return the following day to collect Xpert results. After the initial 2-day visit, study staff visited the health centers every month to reinforce training messages and adherence to the intervention strategy.SIMPLE TB strategy components were introduced at each health center during a 2-day site visit conducted by research staff and NTLP staff. The health center laboratory was provided with a Primo Star iLED microscope , and laboratory officers were trained to perform LED FM using the Uganda National Tuberculosis Reference Laboratory (NTRL) training manual. Proficiency in LED FM was checked by completion of a post-training evaluation form and by practical examination by senior laboratory technologists who are members of the research team. During the study period, EQA for LED FM was performed on a monthly basis by study staff and on a quarterly basis by Uganda NTRL staff. A meeting was held with clinical, laboratory and pharmacy staff to discuss redesigning the TB microscopy process to facilitate same-day diagnosis and treatment of smear-positive TB. Key process changes included screening for cough at the time of patient registration, immediate referral of patients with chronic cough to the laboratory for sputum collection before the clinician visit, on-demand processing of sputum to prepare and analyze two smears rather than batch processing, and training additional staff to initiate TB treatment rather than relying on a single TB pharmacist . TranspoWe extracted patient demographic and clinical data monthly from routine health center records, including laboratory and treatment registers, microscopy and Xpert requisition forms, and GeneXpert software. Individual patient records were matched across data sources manually. Discrepancies were resolved through discussion with health center staff. De-identified patient data were entered into a secure REDCap  database.Outcomes for the present study were based on process metrics that reflect implementation of the SIMPLE TB algorithm components: numbers and proportions of patients referred for TB testing who had two smears examined from the spot specimen, smear-positive patients who initiated anti-tuberculosis treatment, smear-negative patients who had their sputum sample referred for Xpert testing, and Xpert-positive patients who initiated anti-tuberculosis treatment. Additional outcomes included the time-to-diagnosis and time-to-treatment of smear-positive and Xpert-positive patients.2 test of proportions. We performed all analyses using Stata, version 14 .We described patient characteristics and the fidelity of process implementation using proportions with 95% confidence intervals (CIs) for dichotomous outcomes and either medians with interquartile ranges (IQRs) or means with standard deviations (SDs) for continuous outcomes. We compared differences in proportions across sites using the χOf 1212 adults  who undeOverall, 157 of 1212 (13%) patients had microbiologically confirmed  TB . Site-spOf 157 patients with bacteriologically positive TB, 142 (90.4%) were initiated on anti-tuberculosis treatment : 107/116Two smears were prepared and examined from the spot sputum specimens of 1207 (99.6%) patients . Of the P = 0.24). For smear-negative patients, sputum was transported to the Xpert testing site within 1 clinic day for 77–88% of smear-negative patients (P = 0.002) and 17–67% of smear-positive patients with documented HIV infection (P = 0.28). Anti-tuberculosis treatment was initiated at the initial health center visit for 16–73% of smear-positive patients (P = 0.01) and initiated within 2 weeks for 60–83% of smear-negative, Xpert-positive patients (P = 0.56).There was some variability in adherence to the SIMPLE TB algorithm across sites . CompariTo reduce the burden of TB, strategies are needed to facilitate the uptake of new diagnostics and ensure that patients diagnosed with TB are linked to treatment. In this pilot study, we evaluated a streamlined TB diagnostic strategy that sought to reduce the burden of diagnostic evaluations for TB on patients and providers. We found that single-sample LED FM and daily transport of smear-negative samples to Xpert testing facilities are feasible in a resource-limited setting with high TB prevalence. Nearly all patients had two smears examined, and 91% of smear-negative patients underwent sputum testing using Xpert. In addition, Xpert testing had considerable incremental diagnostic yield, contributing to one quarter of all confirmed TB diagnoses. However, even with a streamlined testing process, 10% of patients with bacteriologically positive TB were not initiated on treatment, including 15% of patients diagnosed based on Xpert results. Additional strategies are needed to further reduce pre-treatment loss to follow-up (LTFU).Our findings highlight the continued importance of sputum smear microscopy at peripheral health centers where on-site Xpert testing is currently not feasible. Sputum smear examination of a single spot specimen identified 74% of all patients with bacteriologically positive TB and led to more rapid and consistent linkage to treatment than occurred even with daily referral of samples for Xpert testing. The incremental yield of examining a second smear from the spot specimen was only 2%. This is similar to the 3% incremental yield reported previously in Uganda for single-specimen microscopy,24boda boda was feasible, and the incremental yield of Xpert testing was 26%—more than twice that reported when performing smear examination of a second sputum specimen in even the most favorable evaluation.In settings with access to Xpert testing, our results suggest that examination of a second smear—from the same specimen or from a second specimen—may not be necessary before referring for Xpert testing. Daily transportation of sputum samples to Xpert testing sites by Our streamlined diagnostic strategy also demonstrated the potential to reduce pre-treatment LTFU. At peripheral health centers in Uganda using a routine TB diagnostic process, we found a pre-treatment LTFU rate of 24% in smear-positive patientsBecause of the pilot nature of this study, there were several potential limitations. First, it was not possible to definitively attribute the improvements in processes that were observed to our intervention. A randomized trial that includes a control group is needed to confirm these findings. Second, pre-treatment LTFU remained relatively high despite a streamlined diagnostic process. This is consistent with increasing evidence that guideline implementation requires multifaceted strategies to change behavior.A streamlined diagnostic process that involves the collection of a single sputum specimen followed by on-site examination of one smear and daily referral of smear-negative specimens for Xpert testing is feasible and has strong potential to improve TB diagnosis and treatment at peripheral health centers. This approach has a higher diagnostic yield and lower pre-treatment LTFU than that reported for standard TB diagnostic evaluation. Programs should consider future operational research of this approach while awaiting the results of randomized trials.


Anticonvulsant drugs can cause various forms of skin drug reactions, ranging from exanthema to severe blistering reactions. An association between HLA-B*1502 allele and severe skin reactions have been reported.Fifteen patients with severe skin reactions following treatment with anticonvulsant drugs  and 15 controls (age-matched epileptic patients taking similar anticonvulsants without drug eruption) were included. They were referred to Mofid Children’s Hospital in Tehran, Iran, between Jan 2012 to Jan 2014. Genomic DNA was extracted from peripheral blood of all patients and HLA- B*1502 genotype was detected by real-time PCR.None of the patients was positive for HLA- B*1502, but two patients in control group had positive HLA- B*1502.The HLA- B*1502 is not correlated with severe anticonvulsant drugs -induced skin reactions in Iranian children. Epilepsy is one of the most common serious neurological disorders and the cornerstone of its treatment is usage of anti-epileptic drugs. A minority of patients under treatment with anticonvulsant drugs shows broad-spectrum skin reactions as an adverse effect. These reactions include Stevens–Johnson syndrome (SJS), toxic epidermal necrolysis (TEN) and drug reaction with eosinophilia and systemic symptoms (DRESS), classified according to skin manifestations and percentage of body surface involved) In some Asian and European countries, HLA- B*1502 and HLA-A*3101 has been identified as predictive genetic markers for carbamazepine hypersensitivity .Recent studies have shown an association between HLA-B*1502 and antiepileptic drug-induced cutaneous reactions, including carbamazepine, phenytoin, and lamotrigine .These reactions have high morbidity and can be lethal. Screening of children, who are genetically vulnerable to these skin reactions, can help us to prevent these complications. As there is no data about the relationship of HLA- B*1502 and severe drug reactions to anti-epileptic drugs in Iranian children, we designed this study to elucidate the importance of HLA- B*1502 in adverse reactions.This cross-sectional descriptive study was performed on 15 patients, referred to Mofid Children’s Hospital in Tehran, Iran, between Jan 2012 to Jan 2014, with severe skin reactions  after anticonvulsant drug consumption  and compared to 15 patients with seizure disorders, treated with similar anticonvulsant drugs for at least 3 months and had not shown any adverse reaction (control group). The case and control groups were age and sex matched. The patients’ data were recorded in a questionnaire including age, gender, type of reaction, type of the anti-epileptic drug used, dosage and duration of drug consumption, history of gastrointestinal and respiratory hypersensitivities, concomitant symptoms such as fever and lymphadenopathy, type of seizure including EEG findings, Brain MRI and laboratory findings . A clinical immunologistallergist, based on skin reaction criteria , confirmAfter signing an informed consent by the patients’ parents, 3 mL EDTA blood was taken from patients in case and control groups. The study was approved by Ethics Committee of the hospital. Genomic DNA was extracted from peripheral blood of all patients and control groups, using QIMamp DNA mini kit , according to the manufacturer’s instruction.HLA- B*1502 genotype was detected by real-time PCR (RT-PCR). For this purpose, PG 1502 DNA detection kit  was used.Amplification and detection was done according to the manufacturer’s instruction for applied Biosystems stepone plus.Fifteen patients  with severe skin drug reactions due to anticonvulsant therapy were included in this study. All of the patients were under treatment with aromatic cyclic anticonvulsants  and had shown one of the severe skin drug reactions. The mean and standard deviation of age at presentation were 4.24±2.52 years. The youngest patient was 8 months old and the oldest was 8 years old. The average time interval between drug consumption and drug reaction was 14.87±5.85 days. Eight patients had an abnormal EEG; one was mental retard; seven patients had respiratory or gastrointestinal allergies or positive family history of allergy.Skin drug reactions were seen in 8 patients following phenobarbital consumption, in 5 patients for carbamazepine, in 1 patient for primidone, and 1 patient for lamotrigine. Skin drug reactions were seen in 6 patients as Stevens-Johnson syndrome, in 5 patients as toxic epidermal necrolysis, in one case as overlap syndrome and in 3 patients as DRESS. The number of different reactions following consumption of each drug is summarized in At the time of skin involvement, 10 patients were febrile and 4 patients had lymphadenopathy. Four patients had simple febrile convulsion, 2 had complex febrile convulsion  and 2 patients had one afebrile seizure . Anticonvulsant drugs was discontinued in these patients after skin drug reaction. Seven other patients had recurrent seizures with necessity to continue anticonvulsant drugs.The average dose of drug consumption in patients with skin reaction was 5mg/kg for phenobarbital and 13 mg/kg for carbamazepine. Brain MRI was done in 10 patients, reported abnormal in 2 of them  The HLA- B*1502 allele was tested in all of the patients in case and control groups. This test was negative in all patients in case group, but two patients in control group were positive for HLA- B*1502.Anticonvulsant drugs are a group of pharmacological agents used in the treatment of seizures. Some patients develop hypersensitivity reactions following administration of these drugs , 2. A stMany studies have emphasized the association between antiepileptic drug consumption and skin reactions -12. In oThe average dose of drug consumption in patients with skin reaction was 5 mg/kg for phenobarbital and 13 mg/ kg for carbamazepine. These doses are not considered as a high level and these reactions are idiosyncratic. A strong association were demonstrated between HLA-B*1502 and carbamazepine-induced SJS in Asian areas including Taiwan, Hongkong, and Thailand . The association between carbamazepine-induced side effect and HLA-B*1502 allele were reported in Han Chinese of southern China. The HLA-B*1502 allele frequency in patients with SJS/TEN was significantly higher than controls and HLA-B*1502 allele had 45% positive predictive value, 100% sensitivity, 86.25% specificity and 100% negative predictive value for prediction of skin reactions.HLA-B*1502 allele was seen in all of the patients with SJS due to Carbamazepine usage, but in 9% of the control population . The assStudies in Caucasians patients have reported no association between carbamazepine-induced SJS and HLA-B*1502, thus HLA-B*1502 allele did not exist in all ethnicities .Some studies have implicated that there is an association between the HLA B*1502 as a marker for carbamazepineinduced SJS and TEN in Han Chinese, but there is no published data to show the association of HLA-B*1502 and carbamazepine-induced SJS and TEN in non-Chinese Asians. An association was shown between HLA-B*1502 and cutaneous reactions following usage of other antiepileptic drugs like phenytoin, and lamotrigine .We did not find any association between HLA-B*1502 and antiepileptic drug-induced cutaneous reactions to carbamazepine, lamotrigine, phenobarbital, primidone. Because of high incidence of HLA-B*1502 in many Asian populations, FDA has recommended HLA-B*1502 testing for all Asians before starting carbamazepine treatment . AlthougIn conclusion, no association between HLA- B*1502 and severe skin reactions due to aromatic anticonvulsant drug consumption was noticed. Besides, HLA- B*1502 testing may not be helpful in prediction of severe skin reactions in Iranian population. However, another study with more samples is required to conclude precisely. Tonekaboni Sh: Study design, sampling, revising of the manuscriptJafari N: Study design, sampling, drafting of the manuscriptMansouri M , Jabbehdari S: Study design, sampling Eftekhari R: laboratory testsChavoshzadeh Z: Study design, sampling,Abdollah Gorji F: Statistical analysisMesdaghi M: Study design, sampling, laboratory tests, revising of the manuscriptAll authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.


Correct measurement and continuous monitoring of exclusive breastfeeding are essential to promote exclusive breastfeeding. Measuring exclusive breastfeeding is a complex issue as rates can vary according to the definition, measurement period, questions asked, and infant’s age. This article reviewed the methodology of reporting exclusive breastfeeding in Nepal, and compared exclusive breastfeeding rates using data from a cohort study undertaken in western Nepal.A literature review was first conducted on studies published during 2000–2014. In our cohort study, 735 mother-infant pairs were recruited within the first month postpartum and followed up during the fourth and sixth months.The majority of studies in Nepal, including national surveys, used the World Health Organization (WHO) recommended definition (only breastmilk with the exception of medicine and vitamin syrup), and the most common measurement period was a 24-h recall. Our data demonstrated that the exclusive breastfeeding rate during the sixth month was 8.9% using the recall-since-birth method but was 18.7% using the 24-h recall method. Substantial differences in rates were also found during the first (66.3% vs 83.9%) and fourth months (39.2% vs 61.1%).We found that recent studies reporting exclusive breastfeeding in Nepal varied considerably in methodology. The most commonly used measurement, the 24-h recall, leads to over-estimation of the prevalence of exclusive breastfeeding when compared to the recall-since-birth method. A common standard of reporting exclusive breastfeeding is clearly needed for evidence-based decision making.The online version of this article (doi:10.1186/s12884-016-1180-9) contains supplementary material, which is available to authorized users. Exclusive breastfeeding offers many short and long-term health and nutrition benefits. In the short term, it is the best source of nutrition, and supports optimum growth and development of the infant . In the Comprehensive implementation plan on maternal, infant and young child nutrition was endorsed in 2012 and identified six targets to reduce nutrition-related mortality and mortality [The World Health Organization's (WHO) ortality . Of thesortality . ‘This wortality , p. 9). Measuring exclusive breastfeeding however, is a complex issue because the rate can vary with respect to the recall duration, questions asked, age of infant and definition adopted –12. WHO One of the major issues with measuring exclusive breastfeeding is accounting for prelacteal feeding, which is common practice in countries in South Asia, , 17 wherThe prevalence of exclusive breastfeeding also can vary widely depending on the indicator measure used. The indicator favoured by the WHO employs the 24-h recall methodology to determine the proportion of infants 0–5 months of age who received only breastmilk during the previous day . This meOne of the strategies to reduce under nutrition in Nepal is promotion of exclusive breastfeeding for the first 6 months . MonitorOur paper presents findings from two different methods; a literature review and a large community-based prospective cohort study. Firstly, we conducted a literature review using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Guidelines . A comprIndicators for assessing infant and young child feeding practices part 2: measurement [Secondly, we used data from a prospective cohort study of infant feeding practices conducted during January-October 2014 in the Rupandehi district of western Nepal. This district is located in the south-western plain areas (Terai) of Nepal bordering India in the south. The methodology of this study has been described previously , 32 but surement . Pre-tesWe defined exclusive breastfeeding within the last 24-h as: mother did not introduce any food item besides her breastmilk or other women’s milk .A mother was classified as exclusive breastfeeding during the first month using recall-since-birth if (1) she did not introduce any prelacteal feeds (2) responded that she had been feeding only breastmilk since birth and (3) was exclusively breastfeeding based on 24-h recall. A mothers was classified as exclusively breastfeeding during the fourth month using recall-since-birth if (1) she had exclusively breastfed during the first month, (2) did not introduce any complementary food since the last interview (confirmed reading list) and (3) did not introduce any complementary food in the last 24-h (confirmed reading list). Similarly, a mothers would be classified as exclusive breastfeeding during the sixth month based on recall-since-birth if (1) she had been exclusively breastfeeding during the fourth month, (2) did not introduce any complementary food since last interview (confirmed reading list) and (3) did not introduce any complementary food in the last 24-h (confirmed reading list). Any mother who was not breastfeeding at the time of interview was classified as not exclusively breastfeeding.Ethics approval was obtained from the Human Research Ethics Committee of Curtin University (HR 184/2013) and the Nepal Health Research Council. Mothers provided written consent for themselves and their infants, and personal identifiers were removed before analysis. When mother could not read and write, enumerators read the consent form to them. Mothers provided a thumb print as their initials if they agreed to participate. This method was approved by both ethics committees.Our literature review found only 9 articles which reported exclusive breastfeeding in Nepal. In addition, three NDHS, and one MICS were subsequently included. On reviewing the articles in full, it was found that two studies , 35 had Four national surveys (NDHS and MICS) –27, 30, There were variations in the indicator measure or methods of reporting exclusive breastfeeding used to estimate prevalence of exclusive breastfeeding. Six studies used the 24-h recall measure , 36, 41.th month. On the other hand, the study conducted by Subedi et al. [Age of infant is another important aspect as younger infants are more likely to be breastfed exclusively than their older counterparts. Whilst most studies reported on infants aged 0–5 months , 36, 41,i et al.  reportedi et al.  consideri et al.  reportedIn view of the above methodological discrepancies, we found large variations in the rates of exclusive breastfeeding between studies. With the exception of one prospective cohort study which observed a low rate (29.7%) of exclusive breastfeeding at 6 months (22 weeks) using recall-since-birth , other sWe used data from our cohort study to compare exclusive breastfeeding rates during the first, fourth, and sixth months Fig. ; resultsThis study reviewed and compared the definition and methods of reporting exclusive breastfeeding in Nepal. We found three main issues related to the measurement and reporting of exclusive breastfeeding namely, inconsistent definition of exclusive breastfeeding, indicator measurement and interpretation of the WHO indicator based on 24-h recall.Data collected from the same participants in our cohort study were used to demonstrate the differences in exclusive breastfeeding rates between methods. Our findings suggest that the extensive use of NDHS data based on 24-h recall would inevitably over-estimate the prevalence of lifelong exclusive breastfeeding. In other words, estimates of exclusive breastfeeding rates in Nepal are probably much lower than those previously reported. Such differences in rates between recall-since-birth and 24-h recall have also been demonstrated by others , 22, 23.n = 735), 4 (n = 715), and 6 (n = 711) months), i.e. 2,161 infants in total available for interview from Table In surveys such as NDHS and MICS, age group of infants is another concern when 0–5 month old infants are aggregated to be the denominator for the 24-h recall prevalence. In our cohort study, exclusive breastfeeding during the sixth month was 18.7% by 24-h recall. For illustration purposes, let us regard the study as a cross-sectional survey that had similar number of infants in three age groups: (1 (d 6 n = 7 months),Traditionally the use of prelacteal feeds has been a common practice in Nepal, and more recently the use of formula feeding is becoming increasingly prevalent . PreviouFurthermore, although most studies in the past claimed to adhere to the WHO definition of exclusive breastfeeding , 14, we For health policy advocacy and planning, consistency in definition of exclusive breastfeeding and the measurement indicator is necessary in order to monitor and compare the changes in exclusive breastfeeding rates over time, across regions and between population subgroups. In addition, future research in breastfeeding needs to provide the evidence that aligns with the target of the Ministry of Health Nepal  and the The 24-h recall method is commonly adopted because of its feasibility in large nationally representative studies for evidence-based decision making in developing countries including Nepal , 25, 46 This study found that the use of the 24-h-recall measurement indicator significantly over-estimates life-long exclusive breastfeeding rates. To facilitate uniform and accurate reporting of exclusive breastfeeding rates and monitor national targets for breastfeeding, future efforts should be on reporting exclusive breastfeeding based on the recall-since-birth method, using a cohort study design and repeated measurement to collect infant feeding information.


Falls and fall-related fractures are a major public health problem among the older adults. Although objective measures of poor vision have been reported to be associated with falls, the association of self-reported visual function and vision-specific quality of life (QOL) with falls has been inconsistent across several studies. We investigated the association of self-reported visual function and vision specific QOL with falls in community-dwelling older adults.We conducted a cross-sectional analysis using the baseline data from participants of the Locomotive Syndrome and Health Outcome in Aizu Cohort Study (LOHAS), which is an ongoing population-based cohort study to evaluate the association of physical dysfunction with the clinical outcomes in community-dwelling people. In the present study, the participants aged over 65 years in 2010 were eligible. The exposure variable was the composite score of the VFQ-J11, which was newly developed using item response theory to evaluate vision specific QOL, and the self-reported outcomes were any fall and frequent falls (≥2) over a 1-month period. We estimated odds ratios using separate logistic regression models adjusted for relevant confounding factors.Among 1624 participants, the median (interquartile range) composite score of VFQ-J11 was 86.8 (76.0–95.9). Any fall and frequent falls were reported by 13.9% and 5.4% of participants, respectively. The composite score of the VFQ-J11 was significantly associated with both frequent falls  and any fall .We found that the composite score of the VFQ-J11 was associated with falls in community-dwelling older adults. Detecting individuals with visual impairments associated with falls using the VFQ-J11 and improvement in the score by interventions could prevent falls. We may consider adding self-reported visual function and vision-specific QOL to conventional risk factors for fall among older adults. Falls and fall-related fractures are a major public health problem, especially among older adults. Reportedly, 30% of community-dwelling older adults older than 65 years fall at least once annually, with 5% of falls resulting in fractures and 10% of falls resulting in other serious injury. While tAlthough self-reporting of visual dysfunction might be a useful means of detecting affected individuals, the association between self-reported visual function and falls has been inconsistent across several studies, with several studies reporting a significant association between visual impairment and falls,9 and otst eye surgery, 12.97 (15.37) after the 2nd eye surgery, and 22.16 (19.11) after surgery in both eyes. However, it is also unclear how these changes in VFQ-J11 score affect the risk of falls in individual subjects.The National Eye Institute Visual Function Questionnaire (NEI-VFQ) was developed to measure vision-targeted quality of life (QOL) on the basis that objective clinical measures, such as visual acuity and visual field, cannot fully capture how activities of daily living are affected by vision impairment. SuzukamHere, we investigated the association between the VFQ-J11 and falls in a cross-sectional study. We evaluated how VFQ-J11 scores are related to falls and how useful the VFQ-J11 is in detecting individuals with visual impairment associated with falls.The Locomotive Syndrome and Health Outcomes in Aizu Cohort Study (LOHAS) is an ongoing population-based cohort study which is evaluating the association of physical dysfunction with the clinical outcomes, such as cardiovascular disease, QOL, medical costs, and mortality. In LOHAS, physical function and health-related QOL, including not only objective metrics by physical examinations but subjective metrics using self-administered questionnaires were measured at the baseline evaluation between 2008 and 2010, which was linked with annual health check-ups conducted in the local municipalities. Especially, the unique measurement items including symptoms of overactive bladder and vision-specific QOL, which had not been installed in annual health check-ups, were measured in 2010. The participants of the LOHAS comprised the residents of two municipalities in Fukushima Prefecture, Japan, aged over 40 years, receiving regular health check-ups conducted by local government annually. There were no exclusion criteria. The participants were planned for follow-up of 10 years, linked with the administrative data offered by the municipalities, including the medical receipts and death certificate, to evaluate the clinical relevant outcomes. The design of the LOHAS has been described in detail elsewhere . In the In the present study, eligible participants were aged ≥65 years old. Participants with missing exposure and outcome data were excluded from the final analyses. Missing exposure was defined as not answering all items in the VFQ-J11.The main exposure variable was the composite score of the VFQ-J11. The question items included in the VFQ-J11 were shown in The primary outcome was frequent (≥2) falls over the preceding 1-month period, and the secondary outcome was any fall over the same period. To define fall history, the participants were asked the question, ‘Over the past year, have you fallen down?’, with the response options of ‘Yes’ or ‘No’. The participants who responded ‘Yes’ were then asked the following question: ‘How many times have you fallen down over the past month?’, with the response options of ‘Zero’, ‘Once’, ‘Twice’ or ‘Three or more times’. Those who responded at least ‘Twice’ were defined as experiencing the primary outcome. Those who responded at least ‘Once’ were defined as experiencing the secondary outcome.Baseline characteristics were presented using standard descriptive statistics: medians (interquartile ranges) for continuous variables and percentages for categorical variables. For the primary analysis, odds ratios (ORs) and 95% confidence intervals (95% CIs) for risk of fall were estimated using the multiple logistic regression model. In the logistic regression model, we adjusted for clinically relevant confounding factors: age, gender, presence of diabetes mellitus  and hypWe defined the subscale score in the VFQ-J11 as the average score within the same domains. For example, to calculate the subscale of near vision, which includes three items, the three item scores were added and divided by 3. We examined the association between each subscale and the outcome in separate logistic models which estimated ORs and 95% CIs for frequent falls and any fall.To minimize the selection bias caused by excluding participants who did not complete the VFQ-J11questionnaire, we conducted sensitivity analyses. We performed the analyses with multiple imputation for missing VFQ-J11 data among the participants, after excluding those younger than 65 years and those with outcome values missing. These sensitivity analyses were performed for the same comparisons and outcomes as the primary analyses.LOHAS enrolled 2505 participants in 2010. We excluded those aged <65 years and those with missing data for the exposure and outcome variables, leaving 1624 for inclusion in the primary analysis . The relFrequent falls in the preceding month were experienced by 87 participants. We estimated ORs for frequent falls according to the VFQ-J11 composite score. Odds ratio in the logistic models are shown in Any fall in the preceding month was experienced by 226 participants. We estimated the ORs for any fall according to the VFQ composite score. The logistic models showed that VFQ composite score was significantly associated with any fall as well as frequent falls  .We examined the association of VFQ-J11 subscale scores with frequent falls and any fall in separate logistic models. The ORs are shown in We selected 1726 participants for the sensitivity analyses . The comThis is the first study to investigate the association of VFQ-J11 score with falls. We found significant associations of the VFQ-J11 with both any fall and frequent falls in the previous month. We also found significant associations of three VFQ-J11 subscales  with both any fall and frequent falls in the previous month.There are several possible reasons why the VFQ-J11 composite score is associated with falls in community-dwelling older adults. First, the VFQ-J11 is highly associated with objective parameters of visual function, such as visual acuity and field of vision. The VFQAccording to the subscale analysis, near vision, well-being/distress, and role limitation were associated with both frequent falls and any fall. On the other hand, distant vision was not associated with either frequent falls or any fall. The association with well-being/distress and role limitation was consistent with the results of the development of the VFQ-J11. The queThe present study has several strengths. First, it is the first study to investigate the association between falls and vision-specific QOL retrieved using the validated VFQ-J11 questionnaire. The VFQ-J11 was developed using item response theory and is reported to be more strongly associated with aspects of visual function, such as visual acuity and field of vision, than the VFQ-25. Second,We feel that the findings in this present study have important implications from a clinical and public-health perspective. Although it is already known that objective vision metrics, such as visual acuity and visual field, are associated with falls, actual measurement of these items need special settings and have associated costs, so implementation in all clinical or public-health settings could be difficult. Detecting patients or residents with poor vision and risk of falls using a self-administered questionnaire could be easily implemented in several settings. For example, public-health sectors, such as government institutions, could send their residents the VFQ-J11 questionnaire by mail to effectively detect those with impaired vision-specific QOL and refer them to ophthalmologists.However, the study also has several limitations. First, because of its cross-sectional design, reverse causality was possible. While impaired vision-specific QOL is considered as a plausible cause of falls, reverse causality could be possible in the association of fall with several individual domains of vision-related QOL, such as mental health and functional limitation. Second, the association of vision-specific QOL with falls could not be adjusted by the presence of dementia, which might be a potential confounding factor. Reportedly, individuals with dementia are likely to have difficultly completing self-reported questionnaires by themselves. This stIn conclusion, we found that the composite score of the VFQ-J11 was associated with falls. We may consider adding self-reported visual function and vision-specific QOL to conventional risk factors for fall among older adults. This finding has important clinical implications. First, using the self-administered VFQ-J11 questionnaire, we could detect individuals with visual impairment associated with falls. This might in turn lead to population-based interventions to modify visual impairment to prevent falls. Second, improvement in VFQ-J11 scores  might prevent falls. However, further longitudinal studies are needed to investigate the association between change in VFQ-J11 score and falls.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


Previous transcriptome studies of the human endometrium have revealed hundreds of simultaneously up- and down-regulated genes that are involved in endometrial receptivity. However, the overlap between the studies is relatively small, and we are still searching for potential diagnostic biomarkers. Here we perform a meta-analysis of endometrial-receptivity associated genes on 164 endometrial samples  using a robust rank aggregation (RRA) method, followed by enrichment analysis, and regulatory microRNA prediction. We identify a meta-signature of endometrial receptivity involving 57 mRNA genes as putative receptivity markers, where 39 of these we confirm experimentally using RNA-sequencing method in two separate datasets. The meta-signature genes highlight the importance of immune responses, the complement cascade pathway and the involvement of exosomes in mid-secretory endometrial functions. Bioinformatic prediction identifies 348 microRNAs that could regulate 30 endometrial-receptivity associated genes, and we confirm experimentally the decreased expression of 19 microRNAs with 11 corresponding up-regulated meta-signature genes in our validation experiments. The 57 identified meta-signature genes and involved pathways, together with their regulatory microRNAs could serve as promising and sought-after biomarkers of endometrial receptivity, fertility and infertility. Successful embryo implantation depends on synchronization of a viable embryo and receptive endometrium. In fact, inadequate uterine receptivity has been estimated to contribute to one third of implantation failures, whereas the embryo itself is responsible for two thirds of them3. In assisted reproductive technologies where good-quality embryos are transferred as a standard of care, implantation failure remains an unsolved obstacle6. In patients with recurrent implantation failure (RIF) temporal displacement of the WOI has been described in one out of four patients7, thus suggesting the possibility of these women suffering RIF of endometrial origin. Further, impaired decidualization of endometrial stromal cells that predisposes to late implantation may negate endometrial ‘embryo quality control’ and cause early pregnancy failure10. Hence, better understanding of endometrial receptivity and the importance of the mechanisms involved in mid-secretory endometrial functions is warranted.The period of endometrial receptivity, also known as the window of implantation (WOI), is the limited time (one to two days) when luminal epithelium is favourable for embryo adhesion as the first step of implantation12 to the new ‘omics’ technologies, extensive efforts have been made to understand and characterise receptive endometrium. Traditional endometrial dating criteria, like tissue histology, are obsolete, since their accuracy, reproducibility and functional relevance have been questioned in various randomised studies14. This has encouraged further investigation and application of new technologies to diagnose endometrial receptivity objectively, since reliable diagnostic markers are still lacking and the molecular mechanisms remain largely unclear17.From the first histological dating methods23.With the ‘omics’ revolution, the quest for the transcriptomic signature of human endometrial receptivity has revealed hundreds of simultaneously up- and down-regulated genes implicated in the phenomenon  method29 were not included in the final analysis, since the data on lists of differentially expressed genes were not available publicly nor in response to requests to the authors. A detailed description of the studies included in the final analysis is presented in Table The search process and results of the systematic literature review are presented in detail in Supplementary Figure vs. ‘pre-receptive’ endometrium  for analysis of biological processes and pathways connected to the meta-signature of mid-secretory endometrium. A significant proportion of the genes were involved in biological processes such as responses to external stimuli, responses to wounding, inflammatory responses, negative regulation of coagulation, humoral immune responses, and immunoglobulin-mediated immune responses, among others. Fig. CRABP2, EDN3, OLFM1, SFRP4) in the mid-secretory endometria , which might be due to their low expression levels. The filtering was applied to rule out transcripts with very low or inconsistent expression levels across individuals.Meta-analysis identified 57 genes differentially expressed between the ‘pre-receptive’ and mid-secretory endometrium, where 52 genes were up- and five were down-regulated at WOI. Our RNA-sequencing (RNA-seq) analysis on 20 independent endometrial biopsy samples from fertile women confirmed the differential expression of 52 meta-signature genes  – 48 of these genes were likewise up-regulated and four were down-regulated  in the receptive phase in those cell populations  -sorted endometrial epithelial and stromal cells from two time points in the menstrual cycle, early secretory DDX52, DYNLT3, C1R and APOD expression levels in the receptive phase endometrial samples was confirmed , mined from publicly available AGO-CLIP datasets. Out of 1,403 intersected potential binding sites, 395 showed overlap with experimentally determined Argonaute binding site in human cell lines, filtering down to the most probable microRNA and mRNA interactions. These 395 sites included interactions between 30 genes from our original meta-signature gene list and 348 microRNAs , five target genes  and 10 microRNAs remained important in silico data-mining studies32 using previously published gene expression data have been published to date. Bhagwat et al. created a Human Gene Expression Endometrial Receptivity database (HGEx-ERdb) of 19,285 genes expressed in human endometrium, among which they identified 179 receptivity-associated genes32. Zhang et al. analysed raw data from three previous microarray studies35 and proposed 148 potential biomarkers of receptive endometrium17, while Tapia et al. integrated gene lists from seven previous microarray studies and presented a list of 61 endometrial receptivity biomarkers31. These three in silico analysis studies share only nine genes in common, highlighting the differences not only in in silico analysis approaches applied but also the great variation in study designs, analysis methods and data processing in published transcriptome studies. Clearly the mass of data generated within endometrial transcriptomics studies is under-explored, challenging investigators in future to analyse huge sets of data simultaneously in order to raise power, credibility and reliability of the findings.The human endometrial transcriptome has been extensively studied in the past decade in a search of identifying diagnostic markers of receptive endometrium and to provide more understanding into the complex regulation of endometrial functions. Despite of the mass ‘omics’ data generated, only three 36. In order to overcome these limitations, we directly analysed lists of differentially expressed genes from nine published studies involving a total of 164 endometrial biopsy samples from healthy women. Using a method that has been specifically designed for comparison of gene lists and identification of commonly overlapping genes in various studies, including recently published transcriptome studies in different ethnic groups38, we hope to provide an up-to-date meta-signature of endometrial receptivity biomarkers. Nevertheless, we have to bear in mind that with our approach, analysing the significantly differentially expressed gene lists, we could have missed the potential biomarker genes that were below statistical significance in individual studies but could become relevant in a meta-analysis.The preferred method for gene expression meta-analysis requires analysis of raw expression datasets. However, such a thorough analysis is often not possible as a result of unavailability of raw data, which is partially the case in our meta-analysis. Variation in the number of gene transcripts known at a given moment together with the technological platform employed makes proper integration of raw datasets complicated. In addition, the limited sample size and noisiness of microarray data have resulted in inconsistency of biological conclusionsSPP1, which was detected in all transcriptome studies that were included in our meta-analysis, together with ANXA4, CLDN4, DPP4, GPX3, MAOA, and PAEP, as they have also been identified as putative biomarkers of endometrial receptivity in the previous data-mining and review studies32.The 57 genes identified in our meta-analysis could serve as the top-priority biomarkers of receptive phase endometrium in humans. Of special interest is 41. It is generally accepted that SPP1 interacts with apically expressed integrins on the luminal endometrial epithelium and embryo trophectoderm to attach the conceptus to the endometrium39. Indeed, our cell type-specific RNA-seq validation analysis of endometrial epithelial and stromal cells demonstrates that SPP1 is up-regulated only in the epithelial cells  and not in the stromal cells in the receptive phase endometrium , apolipoprotein D (APOD) and leptin (LEP) pathways intertwining in a large network of cytokine–cytokine receptor interactions37.Secreted phosphoprotein 1, SPP1, also known as osteopontin, is a secreted extracellular matrix (ECM) protein that binds to different cell-surface integrins to stimulate cell–cell and cell–ECM adhesion and communication see Fig. , which pium Fig. . Dysregu49. In order to provide a hospitable environment for the embryo, the balance should be established between the maternal immune tolerance toward a semi-allogeneic implanting embryo and the protective anti-infectious mechanisms in the receptive-phase uterus49. The innate immune system is the first line of defence, providing an immediate response through its ability to distinguish between ‘infectious non-self’ and ‘non-infectious self’ antigens50. Our meta-analysis highlights the importance of five genes involved in innate immunity, specifically in the complement system in mid-secretory endometrium, i.e. C1R, SERPING1, CD55, C4BPA and CFD, as shown in Fig. 51. This protein has been found to be expressed at decreased levels in the endometria of women with recurrent pregnancy loss with antiphospholipid syndrome52. C4BPA is also suggested to have an embryo-protective role, where increased expression of this inhibitor of complement system activation could reduce the possibility of an uncontrolled complement attack on embryo31. Abnormally decreased levels of C4BPA expression in mid-secretory phase endometrium have been detected among women with endometriosis54, implantation failure55 and unexplained recurrent abortion56.Our meta-signature of mid-secretory endometrium highlights the importance of defence responses, specifically the inflammatory response (now recognized as a type of non-specific immune response), immunoglobulin-mediated immune responses  and the complement (major mediator of innate immunity), and coagulation cascade pathway in receptive-phase endometrium. Immune responses, including the inflammatory response, play important roles in the pre- and peri-implantation period, and the up-regulation of genes involved in immune responses during the mid-secretory phase was corroborated in our meta-analysis and has also been highlighted in several previous studies61. Extracellular vesicles are membrane-bound complexes secreted from cells that act as messengers for cell–cell communication and signalling62. The origin of microvesicles and exosomes from endometrial epithelial cells in mid-secretory endometrium, the involvement of endometrial receptivity genes/proteins in exosomes and the uptake of extracellular vesicles by target blastocyst cells is depicted on Fig. 63. Twenty eight proteins from our endometrial receptivity-associated gene list have been experimentally detected in exosomes in humans (ExoCarta database). Our findings support the role of exosomes in endometrial receptivity and the subsequent embryo implantation, and indicate that further research into functional effects of extracellular vesicles in embryo-endometrium cross-talk is needed. Research on extracellular vesicles is a rapidly evolving and expanding field that could offer new opportunities regarding biomarkers of receptive endometrium and embryo implantation. Especially intriguing is the fact that extracellular vesicles have the potential in the development of non-invasive biomarkers and for thriving novel therapies to increase reproductive success.The finding that a significant proportion of meta-signature genes are located in extracellular regions, including extracellular vesicles/exosomes, is intriguing. It is well known that the luminal epithelium with its extracellular area is the first maternal surface to interact with the trophoblast cells of the implanting embryo, but the involvement of extracellular vesicles in the implantation process is a new phenomenon18. Further, studies on mice demonstrate that microRNAs are important in implantation and pregnancy, and the loss of Dicer  within uterus can compromise fertility64. MicroRNAs are non-coding RNA molecules acting as posttranscriptional regulators of gene expression and operate by either degrading or translationally repressing the target mRNAs65. There are now known over 2,000 annotated microRNAs in the human genome66, and since each microRNA may regulate hundreds of genes, it is estimated that microRNAs collectively regulate one third of genes in the genome67. Our prediction and subsequent validation analyses identified 19 down-regulated microRNAs in the mid-secretory phase endometria that resulted in up-regulation of 11 target mRNAs, because of reduced miRNA-mediated repression. Of special interest are miR-130b-3p and ANXA4, miR-548n and SPP1, miR-548ah-3p, miR-30c-1-3p and EFNA1, miR-30c-1-3p and ARID5B, and miR-449c-5p and DKK1 pairs, where the meta-signature gene was validated in two independent validation analyses. The importance of miR-30 family members, miR-30b and miR-30d, in endometrial receptivity have been highlighted in different studies70, however the changed expression of miR-30c-1 has been detected so far in endometrial cancer patients71. In porcine endometrium the expression of miR-30c has been shown to increase during the gestational days, meaning that at the time of implantation this microRNA has been down-regulated when compared to placentation and mid-gestational times72. The increased expression of miR-130b and miR-449c-5p have been detected in endometrial cancer patients when compared to controls74.The involvement of microRNAs in the mid-secretory endometrial functions has been shown by previous studies15. Hence, the hunt for potentially informative and therapeutic markers of uterine receptivity continues. The era of looking for endometrial receptivity markers at other ‘omics’ levels has begun and it is to be hoped that this will result in further promising results . MeSH terms ‘embryo implantation’, ‘endometrium’ and ‘gene expression’ were used. The reference lists of review articles and relevant studies were hand-searched to identify other potentially eligible studies. No language or any other restrictions were applied. Details of the protocol for this systematic review were registered on PROSPERO and can be accessed at: vs. the ‘pre-receptive’ phase (proliferative and/or early secretory phase) were included for final analysis. Endometrial transcriptome analysis in connection with any pathological condition, such as infertility, endometriosis, adenomyosis and cancer was excluded. In addition, transcriptome analyses focussing on different endometrial tissue-compartments separately were not included in the meta-analysis.Abstracts of all articles identified through the search were read for selection of eligible studies. The full text of each eligible article was carefully evaluated. Only original experimental studies published in English and concerning the endometrial transcriptome (using microarray or RNA-seq techniques) in healthy women in the mid-secretory phase vs. ‘pre-receptive’-phase endometrium were extracted from the publications. Where the gene lists were not available, the authors were contacted directly. In transcriptome array studies, if the probe information for a dataset was available, probes were annotated to corresponding ENTREZ IDs using respective Bioconductor 3.1 Affymetrix probe annotation package, in studies using Affymetrix microarray platform. If probe-level data was not available, gene lists were converted to ENTREZ IDs by using the DAVID Gene ID Conversion Tool (DAVID Bioinformatics Resources). Genes reported in a study by Hu et al. that involved Illumina RNA-seq platform38 were standardised to ENTREZ IDs by using the BiomaRt package and ENSEMBL v.75 database. For one study33, the differentially expressed probe lists were acquired by reanalysing the data stored in the Gene Expression Omnibus under accession number GSE6364. We used GEO2R web tool (http://www.ncbi.nlm.nih.gov/geo/info/geo2r.html) with default options for differential analysis and gene list acquisition . Probes not annotated by ENTREZ IDs were removed from subsequent analyses.The lists of genes differentially expressed in mid-secretory Subsequently, lists of up- and down-regulated genes were ranked by their fold changes. In cases of multiple probes detecting the same gene, only the probe with the largest absolute fold change was used for list construction.24. To assess full gene list sizes, the number of detectable gene ENTREZ IDs was used for each array platform and all ENTREZ IDs from BioMart v.75 were used for the RNA-seq study38. For correcting for multiple testing, we used a strict Bonferroni threshold by multiplying all P-values by the maximal number of elements in all input lists. In our case this involved the data published by Hu et al.38, where the total number of ENTREZ IDs available in ENSEMBL v.75  was used as the total number of tests. All lists used in the analysis reflect expression in the mid-secretory group compared with another group (proliferative or early-secretory phase).The robust rank aggregation algorithm (RRA package v.1.1) was used for meta-analysis of the ranked gene lists77. This software was chosen over other enrichment analysis tools as it is up-to-date (updated in May 2016 to Ensembl 84 and Ensembl Genomes 31) and it provides a compact graphical output. The obtained results were corrected for multiple testing by using the g:Profiler tailor-made algorithm g:SCS, which has been shown to provide a better threshold between significant and non-significant results than (commonly used) Bonferroni correction or the Benjamini-Hochberg false discovery rate76.Enrichment analyses for Gene Ontology (GO) terms and biological pathways (KEGG and Reactome) were carried out by using the g:Profiler web tool (biit.cs.ut.ee/gprofiler/)78 and TargetScan 7.079 precomputed prediction results were downloaded from their respective websites . DIANA microT-CDS utilises ENSEMBL v69 transcriptome and miRBase v18 for the prediction, whereas TargetScan 7.0 uses ENSEMBL v75 and miRBase v2180. miRanda v3.3a binary81 was downloaded from http://www.microrna.org/microrna/getDownloads.do and used for the target prediction with ENSEMBL v75 3′UTRs and miRBase v21 mature sequences as an input. The algorithm was used with default settings: Gap Open Penalty: −9.0, Gap Extend Penalty: −4.0, Score Threshold: 140.0, Energy Threshold: 1.0 kcal/mol and Scaling Parameter: 4.0. miRBase internal IDs were used to standardise microRNA names between different miRBase versions.MicroRNA target prediction was performed using three different algorithms – DIANA microT-CDS, TargetScan 7.0 and miRanda v3.3a. In DIANA microT-CDSin silico target predictions, we used database harbouring experimental data about mammalian microRNA binding sites. Therefore, Argonaute  HITS-CLIP and PAR-CLIP datasets for human cell lines were downloaded from StarBase v.2.082 website in the BED format and overlaid with predicted microRNA target sites (http://starbase.sysu.edu.cn/download.php).For additional support to We validated the meta-signature genes in our two independent sample sets from NOTED project  and SARM project . The studies were approved by the local Research Ethics Committees of the University of Tartu and Instituto Valenciano de Infertilidad. An informed consent was signed by all women who agreed to participate in the study, and all methods were carried out in accordance with relevant guidelines and regulations.The detailed description of the study participants and RNA-seq analysis within NOTED project is described in the Supplementary Material. Briefly, 20 healthy fertile women provided endometrial biopsy samples in the early secretory phase (2 days after the luteinizing hormone (LH) peak, LH+2) and in the mid-secretory phase of the menstrual cycle (LH+8) within the same natural cycle. Total mRNA transcriptome and microRNA profile analysis from the same biopsy samples were performed with the RNA-seq method. Differential expression was tested using the edgeR statistical package. Up-regulation was defined as statistically significantly  higher expression  in the mid-secretory phase samples, whereas down-regulation was defined as statistically significantly lower expression in the mid-secretory samples. The primary RNA-seq data are available in the public database Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/) under accession number GSE98386.83. Differential expression was tested using the edgeR statistical package. Up-regulation was defined as statistically significantly  higher expression  in the mid-secretory phase samples, whereas down-regulation was defined as statistically significantly lower expression in the mid-secretory samples. The primary RNA-seq data are available in the public database Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/) under accession number GSE97929. Detailed protocol of the sample collection, processing, and analysis is described in the Supplementary Material.The other independent set of validation was carried out in additional 16 healthy fertile women within SARM project, where cell type-specific RNA-seq on endometrial samples was performed, with separated epithelial and stromal cells. Briefly, endometrial biopsies were collected from 16 healthy fertile women on two different time points within the natural cycle, LH+2 and LH+8. Single cells from endometrial biopsy samples were separated, epithelial cells were labelled with fluorochrome-conjugated mouse anti-human CD9 monoclonal antibody and stromal cells were simultaneously labelled with fluorochrome-conjugated mouse anti-human CD13 monoclonal antibody, followed by flow cytometric analysis and FACS cell sorting. Bulk-RNA full transcriptome analysis of FACS sorted endometrial epithelial and stromal cells was performed with the RNA-seq method, following the single-cell tagged reverse transcription (STRT) protocol with modificationsDDX52, DYNLT3, C1R and APOD on NOTED and SARM project samples using real-time PCR is described in the Supplementary Material.Further validation of the endometrial receptivity signature genes Supplementary MaterialSupplementarty Table 2Supplementary Table 3