Assessment of severity of liver fibrosis is essential in the management of non-alcoholic fatty liver disease (NAFLD). Second Harmonic Generation (SHG) microscopy is a novel optical tissue imaging system that provides automated quantification of fibrosis based on unique architectural features of collagen. This study aims to develop and validate a SHG-based index for automated staging of liver fibrosis in patients with NAFLD.SHG microscopy was performed on archived liver biopsy specimens from 83 patients with NAFLD. A unique algorithm was developed to identify specific SHG parameters that correlated with fibrosis stage. The accuracy of the algorithm was compared against clinical assessment by experienced liver histopathologists using the Brunt fibrosis staging and further validated using the leave-one-out cross-validation method.Mean age of the study cohort was 51.8 ± 11.7 years, with 41% males. A fibrosis index (SHG B-index) was developed comprising 14 unique SHG-based collagen parameters that correlated with severity of NAFLD fibrosis in a continuous fashion. The SHG B-index had excellent correlation with Brunt fibrosis stage . AUROCs for prediction of Brunt fibrosis stages 1, 2, 3 and 4 were 0.853, 0.967, 0.985 and 0.941 respectively. In the cross-validation analysis, the SHG B-index demonstrated high specificity for diagnosis of all grades of fibrosis. A SHG B-index score of >1.76 had an overall diagnostic accuracy of 98.5% for prediction of presence of bridging fibrosis (Brunt stage ≥3) with sensitivity of 87.5%, specificity 98.0%, positive predictive value 96.6% and negative predictive value 92.6%.The SHG B-index is a unique SHG-based index that provides accurate automated assessment of fibrosis stage in NAFLD patients. Non-alcoholic fatty liver disease (NAFLD) is a spectrum of disorders characterized by excessive accumulation of hepatic fat associated with insulin resistance . The preThe current gold standard for assessing liver fibrosis is with liver biopsy . In the There is thus an unmet need for a robust and accurate automated system of fibrosis assessment that will reduce inter-observer variability in the assessment of NASH fibrosis. In addition, this system should provide a continuous scale of fibrosis assessment that objectively evaluates changes in fibrosis severity. Most therapeutic trials for NASH require demonstration of histological improvement in fibrosis as a hard end-point to demonstrate efficacy. Such an automated fibrosis assessment tool would facilitate wide-scale international multi-center therapeutic trials by providing an efficient and reliable system for assessment of liver fibrosis.Second harmonic generation (SHG) microscopy is a novel tissue imaging system based on non-linear optical microscopy which enables observation of endogenous tissue signals such as Two-Photon Excitation Fluorescence (TPEF) and Second Harmonic Generation (SHG) in unstained tissue samples . TPEF prWe have developed an optical microscopy system that can scan and analyze the SHG properties of collagen in unstained liver tissue specimens. In this study, we aim to develop an algorithm based on SHG properties that are unique to NAFLD fibrosis and to validate its reliability to predict severity of fibrosis in patients with NAFLD.Archived formalin-fixed, paraffin-embedded liver biopsy samples from 83 patients with NAFLD were retrieved for analysis. Subjects were identified from a database of patients in the Department of Gastroenterology and Hepatology of the Singapore General Hospital with a clinical diagnosis of NAFLD who underwent liver biopsy for clinical indications between 2005 and 2015. All subjects fulfilled the definition of NAFLD with >5% hepatic steatosis on liver biopsy in the absence of viral hepatitis, significant alcohol consumption , autoimmune and hereditary liver diseases [™ system . SHG microscopy was used to visualize collagen and two-photon excited fluorescence (TPEF) microscopy was utilized for visualization of the other cell structures. The settings of laser were similar to previous publications [2 instead of scanning the entire biopsy tissue.Unstained tissues of the 83 liver biopsy samples were imaged by the Genesisications . Images ications . Thus, 1Landmark structures such as the portal tract and central vein were marked out on the H&E-stained slide and correlated to various variables on the SHG image . This alDetermining specific regions: The SHG signal and TPEF signal in the background of SHG/TPEF images were recognized as noises, which were firstly removed . Quantitative fibrosis assessment by the SHG B-index was correlated with Brunt fibrosis score using Spearman correlation. AUROC analysis was performed to evaluate the accuracy of SHG B-index for prediction of the different grades of Brunt fibrosis. Optimal cut-off values of the quantitative SHG B-index were identified for prediction of Brunt fibrosis grades. Leave-one-out cross-validation (described above) was used to validate the performance of the SHG B-index model. The sensitivity, specificity, positive and negative likelihood ratios of SHG B-index for prediction for presence of bridging fibrosis (i.e. Brunt fibrosis grade 3) were calculated. Statistical significance level was set at p<0.05.Mean age of the study cohort was 51.8 ± 11.7 years, with 41% males and 77% of Chinese ethnicity. Demographic, anthropometric and biochemistry data are summarized in AUROC analysis was performed to evaluate the accuracy of the SHG B-index to identify the various fibrosis stages . AUROC oOptimal cut-off values of SHG B-index to identify specific Brunt fibrosis groups were calculated using Youden’s index . To assess for presence of fibrosis (i.e. to differentiate between no fibrosis and presence of fibrosis), the optimal cut-off value of the SHG B-index was 1.18. The cut-off values for presence of significant fibrosis, advanced fibrosis and cirrhosis were 1.33, 1.76 and 2.76, respectively.The performance of the SHG B-index was validated using the leave-one-out cross-validation method . OverallUsing the unique capabilities of SHG to identify collagen parameters associated with NAFLD fibrosis, we developed a composite index (the SHG B-index) that has high accuracy for identifying various stages of NAFLD fibrosis. Using a cross-validation method, we confirm that the SHG B-index is reliable for the prediction of severity of liver fibrosis compared to traditional histopathologists’ assessment using Brunt classification. A SHG B-index of >1.76 is highly sensitive and highly specific for presence of bridging fibrosis, a clinically important feature that is associated with poor prognosis in NAFLD patients. This study thus provides evidence to support the potential clinical application of an automated system of fibrosis staging using unstained liver biopsy specimens.The development of SHG-based optical microscopy represents a potential paradigm shift in the histopathological assessment of fibrosis, providing several advantages over conventional histopathological assessment. The unique crystalline triple-helix structure of fibrillar collagen provides a specific SHG signal that allows accurate identification, characterization and quantification of liver fibrosis . This prSHG-based assessment of liver fibrosis has been shown to be reliable in chronic viral hepatitis. Correlation between SHG signal and METAVIR fibrosis score in patients with chronic viral hepatitis was initially described by Gailhouste and colleagues  and subsWe observed that the quantitative distribution of fibrosis assessed by SHG B-index was highly sensitive and specific for the identification of advanced fibrosis in NAFLD but was less discerning in discriminating between early stages of fibrosis. In the early stages of fibrosis (Brunt stage 1 and 2), the amount of collagen in the liver is similar, but the distribution of collagen is different being predominantly peri-sinusoidal in stage 1 and peri-portal in stage 2. However, in advanced fibrosis (Brunt stage 3) we observe a significant increase in the SHG B-index, reflecting an increase in the amount of collagen deposited as the fibrous tissue progresses from the portal tracts to the central veins. The SHG B-index thus provides a reliable method to identify NAFLD patients with advanced fibrosis who are at increased risk of liver-related morbidity and mortality.SHG microscopy has several important clinical implications in the management of NAFLD patients. Firstly, it provides a highly reliable automated assessment of fibrosis that can reduce interobserver variability and improve accuracy of histological staging of liver fibrosis. Secondly, it can be performed on unstained tissues thus avoiding the need for cumbersome staining procedures and degradation artifacts. Thirdly, SHG optical microscopy provides a quantitative assessment of fibrosis on a continuous scale which reflects dynamic change in fibrosis more accurately than the conventional semi-quantitative histological grading systems. With the increase in new anti-fibrotic therapeutic compounds, the use of a quantitative method with a continuous scoring scale to assess progression or regression in severity of fibrosis is intuitively more reflective of the true therapeutic response. The primary utility of SHG-based assessment of liver fibrosis may be in therapeutic NAFLD trials, many of which require serial liver biopsies for assessment of improvement in fibrosis. The automated fibrosis scoring provided by SHG microscopy helps to minimize inter-observer variability and reduces the cost and logistics associated with shipping of samples for central histology review. This automated, standardized and clinically reliable fibrosis assessment system may thus pave the way for future large, multicenter, international collaborative studies.Although SHG optical microscopy offers several novel applications in fibrosis assessment, it is important to emphasize that this technology does not replace the need for invasive liver biopsy and cannot replace the key role of the histopathologist in clinical practice . The infWe acknowledge that our study is limited by a modest sample size from a single center and lack independent external validation. Nonetheless, we have employed a robust methodology and analysis to provide reliable results, which we believe are important to the scientific community. A larger multi-center is currently being planned to validate the SHG B-index in an independent cohort. Another limitation is that this study did not examine changes in fibrosis stage from subjects with repeat liver biopsies. Hence, the role of SHG B-index for assessment of progression or regression of NAFLD fibrosis in longitudinal pre-and post-treatment liver biopsies needs to be validated as well, particularly in the context of response to pharmacological intervention for NAFLD.In conclusion, this study demonstrates that the SHG B-index, a unique fibrosis index based on SHG optical microscopy, provides highly reliable automated assessment of fibrosis in patients with NAFLD. This index has a high specificity for advanced fibrosis which is a clinically important sentinel point in the management of NAFLD.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 FigOne sample is randomly retained as the validation set while the remaining 82 samples are used as the training set to develop a prediction model. The performance of the prediction model is then tested on the single validation sample. The cross-validation process is repeated 83 times, with a different sample left out each time. The overall performance of the prediction model to predict the various fibrosis stages is assessed based on the 83 predicted B-indexes.(PDF)Click here for additional data file.


This article comments on:Rümpler F, Theißen G, Melzer R. 2018.69, 1943–1954A conserved leucine zipper-like motif accounts for strong tetramerization capabilities of SEPALLATA-like MADS-domain transcription factors. Journal of Experimental Botany . MADS family transcription factors are crucial during plant reproductive development, and have evolved a complex protein–protein interaction (PPI) network. Proteins of the SEPALLATA (SEP) clade are required for tetramer formation and can act as critical ‘hubs’ in the network.et al. (2018)Rümpler have now provided quantitative measures of the contribution of individual amino acids to cooperative DNA binding, laying a foundation for predicting MADS tetramer formation based on primary sequence. It is an important step forward in understanding how cooperativity affects processes from flowering time to floral organ identity.E. coli) to those that are more complex like Arabidopsis  clade, and members of this clade are able to act as hubs within the MADS PPI network and drive the formation of distinct tetrameric complexes , B (APETALA3 and PISTILLATA), C (AGAMOUS) and E (SEPALLATA1-4), results in the formation of different floral organ types  shows a structural composite overview of a SEP3 homotetramer with each monomer coloured differently . The C-terminal domain is not shown, as no homologous structures are available for modelling. The structures of the MADS domain from MEF2 (1TQE) and K domain from SEP3 (4OX0) were used to generate the composite model shown. Transparent highlights: DNA binding domains, light yellow boxes; dimerization interfaces, light purple ovals; tetramerization interface, light blue oval.Panel (B) shows a close-up of the dimerization and tetramerization from (A) with the yellow and green monomers transparent for clarity. Labelled residues have been mutated and characterized in terms of DNA binding and/or tetramerization ] resulting in amphipathic helices in which one side is hydrophobic, thus providing an oligomerization surface for a partner amphipathic helix to bind, forming a coiled-coil (The coiled-coil domain of SEP3 responsible for the formation of tetrameric MADS complexes has recently been structurally characterized and consists of two alpha helices (led-coil .In the paper by cis-elements, TF protein–protein binding and recruitment of co-factors. DNA sequence, chromatin structure and concentration of TFs and cofactors all play important roles in this process. Structural studies of the tetramerization domain of SEP3 highlight the residues likely to be important for dimer and tetramer formation (in vitro by SELEX (cis-elements has also been examined through systematic variation of the number of nucleotides between DNA-binding sites (in vivo (Proper gene regulation by MADS TFs requires an intricate balance of binding events: selection of specific in vitro biochemical, structural and biophysical studies of MADS TFs are crucial for understanding how they function in vivo, and predicting the cooperative binding of MADS complexes based on the primary amino acid sequence is an important advance. The research outlined by Rümpler and colleagues lays a foundation for understanding how substitutions at key residues alter cooperative binding and change the MADS protein–protein interactome.Detailed in vitro data now available coupled with ChIP-seq experiments has given us a deeper understanding of the MADS TF–DNA binding landscape. The next step will be to examine in vivo the role of cooperativity in MADS TF function and gene regulation. How important cooperative binding is to gene regulation is an open question in the field and one that still needs to be answered.The wealth of


Despite continual progress in the identification and characterization of trait- and disease-associated variants that disrupt transcription factor (TF)-DNA binding, little is known about the distribution of TF binding deactivating mutations (deMs) in enhancer sequences. Here, we focus on elucidating the mechanism underlying the different densities of deMs in human enhancers.We identify two classes of enhancers based on the density of nucleotides prone to deMs. Firstly, fragile enhancers with abundant deM nucleotides are associated with the immune system and regular cellular maintenance. Secondly, stable enhancers with only a few deM nucleotides are associated with the development and regulation of TFs and are evolutionarily conserved. These two classes of enhancers feature different regulatory programs: the binding sites of pioneer TFs of FOX family are specifically enriched in stable enhancers, while tissue-specific TFs are enriched in fragile enhancers. Moreover, stable enhancers are more tolerant of deMs due to their dominant employment of homotypic TF binding site (TFBS) clusters, as opposed to the larger-extent usage of heterotypic TFBS clusters in fragile enhancers. Notably, the sequence environment and chromatin context of the cognate motif, other than the motif itself, contribute more to the susceptibility to deMs of TF binding.This dichotomy of enhancer activity is conserved across different tissues, has a specific footprint in epigenetic profiles, and argues for a bimodal evolution of gene regulatory programs in vertebrates. Specifically encoded stable enhancers are evolutionarily conserved and associated with development, while differently encoded fragile enhancers are associated with the adaptation of species.The online version of this article (10.1186/s13059-019-1750-z) contains supplementary material, which is available to authorized users. Analysis of genomic variants in humans and model species such as mouse and fruit fly is providing opportunities for understanding the genetic basis of complex traits and disease predisposition. The vast majority of complex trait-associated variants from the Genome-Wide Association Studies (GWAS) are located in non-coding regulatory regions—only approximately 5% of GWAS SNPs are located in the protein-coding regions , 2. AccuTo accurately identify the causal regulatory variation, advanced and flexible machine learning models, such as support vector machine (SVM) , 15 and Employing CAPE to the enhancer regions in hepatocellular carcinoma (HepG2) cells, we observed a large spectrum of density of putative deactivating mutation positions (dubbed deMPs) across the panel of these enhancers. Enhancer deMP nucleotide content ranges from 0.04 to 36%. On average, approximately 1.5% of enhancer positions are deMPs and thus are prone to deactivating effects. This startling difference in deMP density motivated us to investigate the inherent mechanisms of the regulatory programs of the enhancers with different densities of deMPs, such as inter-TF interactions and evolutionary constraints. After categorizing HepG2 enhancers to three sets  based on their densities of deMPs, we observed that the deMP densities are negatively correlated with the functional and evolutionary stability of enhancers. Fragile enhancers have elevated deMP densities that make them prone to a wide range of single-nucleotide deactivation mutations and, thus, make it harder to preserve their function during evolution. This kind of enhancer is specific to the defense system and cellular maintenance and might benefit from the panel of deMPs in them, which might represent a pathway for evolutionary innovation and adaptation. By contrast, stable enhancers are impervious to single-nucleotide deactivations and more functionally conserved. This set of HepG2 enhancers is strongly associated with liver development and regulation of transcription factor activity. In addition, the two sets of enhancers have different cohorts of TFBSs and benefit from different modes of inter-TF interactions. The homotypic TFBS cluster usage provides the stable enhancers with more buffering once a mutation takes place. Additionally, we observed that the mode of TF interaction and multiple layers of chromatin context contribute more to the binding site sensitivity to one single-nucleotide deactivation, as compared to the cognate motif itself.By applying transgenic mouse reporter assay of two stable enhancers in human heart left ventricle, we observed that lacZ expression of the transfected enhancers was abolished (or diminished) in the mouse embryonic hearts due to the top 5% mutations identified by our algorithm, which substantiate the functional impact of the predicted deactivating mutations and further support the rationale of the two classes of enhancer activities.cis-regulatory causal variants and examine their distribution in HepG2 cell line, we applied the SVM classifier CAPE [S)] (see the “S) represent the overall contribution of the binding affinity change and interactions of combinations of TFs within the surrounding sequence, respectively, to the impact on enhancer activity. The mutations identified by CAPE (FPR ≤ 0.01) were dubbed deactivating mutations (deMs), as they are likely to disrupt the binding of an essential TF and lead to a deleterious impact on the enhancer activity and, in some cases, result in enhancer deactivation. The genomic positions holding at least one deM were dubbed deMPs. In general, this approach is limited to mutations that are observed frequently and that have matching expression observations, in the relevant tissue or cell type. For the tissues without eQTL information, this method is not directly applicable. Overall, 167,303 deMPs reside in approximately 57%  of HepG2 active enhancers. We then focused on the active enhancers with at least one deMP in their sequence. On average, approximately 1.5% of enhancer positions are deMPs cis-reguln = 11,831) into three categories based on their deMP density: fragile enhancers , regular enhancers , and stable enhancers   in this study. To investigate the relationship between enhancer fragility and deMP density, we first assessed the evolutionary constraints of deMPs by computing the percentage of the positions different from the chimp genome and the fraction of positions carrying single-nucleotide polymorphisms (SNPs). Overall, deMPs are under stronger purifying selection than regular, non-repetitive enhancer nucleotides , rheumatoid arthritis, and multiple sclerosis. This suggests a dominant role of fragile enhancers in the defense system to a greater extent compared with stable enhancers ] (see the “S). The deMP with the highest CAPE score within a binding site was denoted as max-deMP, and the mutation with the highest CAPE score within a binding site not overlapping deMPs was denoted as max-non-deMP. By sequentially taking the mean of WS(Δ) and WS(S) over the max-deMPs (or max-non-deMPs) across all the binding sites of each TF, we observed that although the binding affinity change caused by the max-deMP is noticeably larger than that caused by max-non-deMP  [http://enhancer.lbl.gov) [To directly test the ability of CAPE to predict the functional consequence of sequence variation on enhancer activity in vivo, we turned to two stable enhancers active in the human heart (left ventricle) see the “” sectionde.org/) ; and (3)lbl.gov)  , but also not all non-coding mutations affecting TF binding have a measurable impact on gene expression. Here, we identified the mutations that are likely to deactivate TF binding and investigated the sensitivity of enhancers to single-nucleotide mutations. The fragile enhancers with abundant deactivating mutation nucleotides are associated with the immune system and regular cellular maintenance, whereas the stable enhancers with only a few deactivating mutation nucleotides are associated with development and regulation of transcription factors and are evolutionarily and functionally conserved. These two classes of enhancers encoded by different regulatory programs have contrasting levels of tolerance of deactivating mutations and employ different modes of TF-TF interactions. We found that TF-TF interactions, and not individual TF-DNA binding events, are the key contributor to enhancer deactivating mutations. Our study profiles deleterious mutations in human enhancers and identifies specific enhancer nucleotides most sensitive to mutations, thus providing an atlas of non-coding mutations likely linked to disease susceptibility and evolutionary innovations. However, versatile binding patterns of different TFs and interaction between TFs of these two classes of enhancers await further elucidation.The general idea of identification of deMP is first to identify the candidate deactivating mutations that disrupt a putative binding site and next to score these candidate deactivating mutations using CAPE, a tool we developed to identify causal regulatory variant in enhancer regions . All thrk-mer vocabularies trained on ChIP-seq enhancers to infer the sequence specificities of TFBSs. The enriched k-mers (k = 8) were assumed to be the potentially functional TFBSs [k-mers in HepG2 enhancers, we first generated a set of controls for each enhancer sequence. Controls were randomly sampled from the whole genome with the same GC-content, repeat-content, and length as the corresponding enhancer sequence. Five control sequences were extracted for each enhancer. In cases when not enough controls with our strict criteria  could be identified, we created additional controls by reshuffling enhancer sequences. For each of the possible 32,896 k-mers (k = 8), we used the Fisher exact test to evaluate the enrichment of k-mers in the HepG2 enhancer set and identified the top 522 k-mers significantly enriched in enhancers (p ≤ 1e−3 after the Bonferroni correction) as potentially functional k-mers and 30,647 insignificant k-mers (p > 1e−3 without the Bonferroni correction) as background k-mers.Specifically, we utilized the al TFBSs  on ChIP-k-mer to a background k-mer once we identified top k-mers in the positive training set. The candidate deactivating mutations were next scored by CAPE. For the HepG2 cell line, only the mutations with significant CAPE scores  were considered to be deMs. We used the change of the associated k-mers to identify the candidate deactivating mutation before applying CAPE due to the limitation of the CAPE score. The output of CAPE is the probability of a mutation being a causal regulatory variation by either decreasing or increasing the enhancer activity. Since we are particularly focused on mutations deactivating enhancers, we need to limit the candidate deactivating mutations to the ones that could disrupt a potential binding site using the k-mer vocabularies.As we did in our previous study , we applTo identify the deMPs in the left ventricle, we trained CAPE on the human left ventricle eQTLs by integrating the regulatory signals of this tissue . We then scored all possible single-nucleotide variants (SNVs) in the left ventricle enhancer region. Only the mutations with CAPE score ≥ 0.58276 (FPR ≤ 0.01) were identified to be deMs  a minimum binomial observed region hits and hypergeometric observed gene hits of 10, and (3) a minimum binomial region and hypergeometric gene set fold enrichment of 2. The −log10 binomial p values were plotted on the y-axis. To show that the GO enrichment of both fragile and stable enhancers is robust by different gene association rules, the other two gene association options  were also applied  version 3.0.0 . In the r2 > 0.8, maximum distance of 500 kb). The enrichment of stable enhancer SNPs coinciding with a disease type relative to fragile enhancer SNPs was evaluated as −logP of the hypergeometric distribution, and vice versa.The NHGRI GWAS Catalog was downloaded in September 2016 [A) in a set of enhancers is determined by formula For the purpose of identifying the location of potential binding sites, we used the profiles of binding sites for vertebrate TFs stored in Jaspar , CIS-BP If an enhancer harbored at least three potential binding sites for a TF expanding no more than 1 kb, we assumed that this enhancer had at least one homotypic TFBS cluster. Analogously, if an enhancer harbored at least three potential binding sites for different TFs expanding no more than 1 kb, we assumed that this enhancer had at least one heterotypic TFBS clusters.S), are the basic component of features for each signal  features. Nk (= 5) is the number of k-mer sizes . NkmerSignature (= 2) is the number of signatures including the binding affinity change of the potential binding site due to the mutation (Δ) and the overall binding capabilities of the nearby sequence context of the genetic variant (S). NsignalTrack is the number of the chromatin data ) Additional file 2:Supplementary tables. (XLSX 37 kb)Additional file 3:Review history. (DOCX 396 kb)


When influenza A virus infects an immune individual, preexisting memory B cell (MBC) activation and rapid anamnestic antibody production plays a key role in viral clearance. The most effective neutralizing antibodies target the antigenically variable head of the viral hemagglutinin (HA); antibodies against the conserved HA stalk provide broader but less potent protection. In this review, we provide a comprehensive picture of an adult’s HA-specific antibody response to influenza virus infection. The process is followed from preexisting HA-specific MBC activation and rapid production of anti-HA antibodies, through to germinal center seeding and adaptation of the response to novel features of the HA. A major focus of the review is the role of competition between preexisting MBCs in determining the character of the HA-reactive antibody response. HA novelty modifies this competition and can shift the response from the immunodominant head to the stalk. We suggest that antibodies resulting from preexisting MBC activation are important regulators of anti-HA antibody production and play a role in positive selection of germinal center B cells reactive to novel HA epitopes. Our review also considers the role of MBCs in the effects of early-life imprinting on HA head- and stalk-specific antibody responses to influenza infection. An understanding of the processes described in this review will guide development of vaccination strategies that provide broadly effective protection. B cell memory generated by influenza A virus (IAV) infection and vaccination consists of antibodies (Abs) and memory B cells (MBCs). Preexisting Abs against the virus’s surface glycoproteins, the hemagglutinin (HA) and neuraminidase, have direct antiviral activity and provide the most effective protection against initiation or progression of infection ,2. If inEach monomeric component of the homotrimeric HA consists of two structurally distinct domains: a membrane-distal head domain containing the receptor binding site and a membrane-proximal stalk domain . Abs agaAbs against the IAV HA are also the basis of “original antigenic sin (OAS)” as originally termed , and morB cell activation and the production of Abs against the HA of an infecting IAV begins with transport of the HA  from the site of infection to local lymphoid tissues and lymph nodes. Antigen binding via the B cell receptor (BCR) and uptake, processing, and presentation to establish cognate interactions with CD4 T cells are essential for MBC and naïve B cell activation ,18. ThisAnalysis of the Ab response to IAV infection has generally focused on measurement of circulating Abs against the HA of a strain likely to represent the infecting virus, such as the strain in the most recent seasonal influenza vaccine. However, recent studies have emphasized that anti-HA Ab production is characterized by increased titers against a chronological range of HA variants ,13,16. WEarly production of anti-HA Abs following IAV infection in immune adults results from activation of preexisting MBCs, a model based on analysis of Ab-secreting plasmablasts (PBs) that appear in the circulation at about the same time as the first virus-induced Abs. These PBs generally reach peak frequencies 4–6 days post-symptom onset, but remain detectable for another 1–2 weeks ,25. MoreThe site of early MBC-derived PB formation in responding lymphoid tissue has often been imprecisely described as “extrafollicular” . RecentlIt is generally accepted that OAS patterns of HA-reactive Ab production after IAV infection reflect the competitive dominance of MBCs reactive to conserved epitopes on the HA head domain, although experimental support for this idea is lacking ,15. The The reactivity profiles of Abs expressed by individual PBs generated early in the response to IAV infection have not been extensively characterized. However, limited studies indicate that at least some of the PB-expressed Abs bind to one or more older HAs, sometimes with higher affinity than to the infecting virus HA ,30. ThesIn addition to the broad HA head-reactive Ab response, approximately 10%–20% of anti-HA Abs generated by seasonal IAV infection bind to the stalk domain. The response to the stalk follows the same kinetics as the anti-head response and includes early induction of stalk-specific PBs with high numbers of mutations in immunoglobulin variable genes, indicating formation from activated MBCs ,26. A keStudies of responses to infection and vaccination with the 2009 pandemic H1N1 (pH1N1) IAV emphasize the importance of competition between MBCs in determining the nature of HA-specific Ab production. The H1 of the pH1N1 IAV was substantially novel to most individuals less than approximately 60 years of age when the virus began circulating in humans. Initial exposure of these individuals to the pH1N1 virus through infection or vaccination generated anti-HA Abs that were broadly cross-reactive with head and stalk epitopes of multiple influenza strains ,35,36. IAn effect of early-life imprinting on the character of an individual’s Ab response to the HA head following IAV infection is well-established , but it The pathways that maintain or expand HA-reactive MBC populations after IAV infection are not fully understood. For instance, it is unclear whether preexisting IgG MBCs activated in the SPF generate both PBs and MBCs via extra-GC pathways or whether MBCs are only generated in GCs after some level of remodeling of antigen reactivity. In the absence of GCs, naïve B cell activation can produce IgG MBCs that have not undergone affinity maturation ,45, raisA subset of HA-reactive B cells activated by IAV infection enter GC reactions where they undergo affinity maturation . A propoAfter Ab V-region somatic hypermutation, GC B cells that express BCRs with high antigen affinity are positively selected for further cell division and mutation or for differentiation along pathways that generate plasma cells or MBCs. The process of positive selection is not fully understood, but signaling to GC B cells via the BCR and CD40 plays a central role. A key step is competition between GC B cells for antigen displayed on the surface of follicular dendritic cells (FDCs); Ag that is bound with sufficient affinity by GC B cells is taken up, processed, and presented to secure cognate help from Tfh cells . ImportaRecent studies of human responses to avian HAs, including H5 expressed by a replicating adenovirus, provide evidence that GC activity continues for many months after immunization ,33. It iPreexisting anti-HA Abs and anti-HA Abs generated at various stages after IAV infection are thought to play an important role in regulating the fine specificity of the anti-HA Ab response . The antOur goal in this review is to combine observations from human and animal studies to provide a picture of the HA-specific B cell response to IAV infection in adults. A critical determinant of the nature of the response in adults is the HA-specific B cell memory established by HA exposure through IAV infection and vaccination over many years. Of central importance is the composition of an individual’s HA-reactive MBC pool, a population that even in adulthood reflects the imprint of early-life HA exposure and is responsible for OAS patterns of Ab production early in the response to IAV infection. PB formation from activated preexisting MBCs with high affinity for the HA of the infecting virus optimizes the protective efficacy of the initial wave of anti-HA Abs. We emphasize MBC competition as a key determinant of the patterns of activation of preexisting MBCs. The novelty of the HA of the infecting virus has a profound influence on the outcome of MBC competition and (for example) determines the strength of Ab production against the HA stalk. Preexisting MBC activation generates cells that along with activated naïve B cells, seed GCs for generation of MBCs and plasma cells adapted to variant/novel HA epitopes. This process involves another form of B cell competition, the competition between GC B cells for antigen held by FDCs; acquisition of this antigen is a prerequisite for positive selection. We suggest that Abs resulting from preexisting MBC activation, more so than preexisting circulating Abs, play an important regulatory role in positive selection through epitope masking. Depending on the epitope specificity of Abs derived from preexisting MBCs, GC B cells will be refocused on the immunodominant HA head or on particular drifted head epitopes. Our review also considers the generation and expansion of MBCs at different points in the response to both maintain OAS response patterns and adapt to emerging HAs. Our increasing understanding of MBC competition, immunodominance hierarchies, and Ab regulation of B cell responses will continue to guide development of vaccine composition and administration strategies to optimize B cell-mediated protection generated against IAV infection ,65,66,67


Proximal humerus fractures are the third most common fracture in the elderly population and are expected to increase due to the aging population. Surgical fixation with locking plate technology has increased over the last decade despite a lack of proven superiority in the literature. Three previous randomized controlled trials have not shown a difference in patient-centered outcomes when comparing non-operative treatment with open reduction and internal fixation. Low patient enrollment and other methodological concerns however limit the generalizability of these conclusions and as a result, management of these fractures remains a controversy. By comparing the functional outcomes of locked plate surgical fixation versus non-operative treatment of displaced three and four-part proximal humerus fractures in the elderly population with a large scale, prospective, multi-centered randomized controlled trial, the optimal management strategy for this common injury may be determined.We will conduct a prospective, single blind randomized controlled parallel arm trial to compare non-operative management of proximal humerus fractures with open reduction and internal fixation using locked plating technology. One-hundred and sixty patients > age 60 with acute 3- or 4- part proximal humerus fractures will be randomized to either open reduction and internal fixation with locked plating technology or non-operative management treatment arms. The primary outcome measure is the Constant Score at 24 months post-operative. Secondary outcome measures include the American Shoulder and Elbow Surgeon’s Score (ASES), EuroQol EQ-5D-5 L Health Questionnaire Score, short form PROMIS upper extremity score and IPAQ for the elderly score. Further outcome measures include assessment of the initial classification, displacement and angulation and the quality of surgical reduction via a standard computed tomography (CT) scan; rates of non-union, malunion, arthrosis, osteopenia or other complications including infection, nerve injury, intra-articular screw penetration, reoperation rates and hospital re-admission rates.The results of this trial will provide Level 1 evidence to guide decision-making in the treatment of proximal humerus fractures in the elderly population.NCT02362100. Registered 5 Feb 2015.ClinicalTrials.gov  Proximal humerus fractures account for 6% of all fractures  and are n = 650)  of the fragments based on post-operative CT scan and the Constant functional outcome measure will be determined in the surgical group using a multivariable regression analysis.In addition to the primary and secondary analyses outlined above, several other analyses are planned, though the study is not primarily powered for these statistical analyses. We will perform a subgroup analysis on the above outcome measures to assess whether there is a difference between 3- and 4- part proximal humerus fractures. This will primarily include a subgroup analysis of the Constant score, within groups of fracture type: 3- and 4- part fractures. The second will be a multivariable assessment of the progression of osteoarthritis between pre- and post-operative shoulders. A multivariable regression analysis will also be conducted of possible factors associated with progression of shoulder osteoarthritis, including demographic variables and fracture type. The third will be a multivariable regression analysis to determine which factors may be associated with functional outcome: with the Constant score as the dependent variable, demographic and radiographic factors (including osteoporosis) will be analyzed to determine if prognostic factors exist that may assist in planning treatment.The optimal management of proximal humeral fractures remains controversial . Three pThe PROFHER trial  is curreFurther potential strengths include the use of pre-operative CT scan for analysis of fracture displacement/angulation, and inclusion of the IPAQ score, which is tailored towards the elderly population in order to assess the functional demands of the population within the study and how this impacts performance on the other functional assessment tools.Our data will provide level 1 evidence that will inform the management of proximal humerus fractures in the elderly population which in turn will allow surgeons to recommend the most effective treatment for patients with this injury while taking imaging characteristics into account.


This study aimed at evaluating the cost-effectiveness of different non-invasive imaging-guided strategies for the diagnosis of obstructive coronary artery disease (CAD) in a European population of patients from the Evaluation of Integrated Cardiac Imaging in Ischemic Heart Disease (EVINCI) study.Cost-effectiveness analysis was performed in 350 patients  with symptoms of suspected stable CAD undergoing computed tomography coronary angiography (CTCA) and at least one cardiac imaging stress-test prior to invasive coronary angiography (ICA) and in whom imaging exams were analysed at dedicated core laboratories. Stand-alone stress-tests or combined non-invasive strategies, when the first exam was uncertain, were compared. The diagnostic end-point was obstructive CAD defined as > 50% stenosis at quantitative ICA in the left main or at least one major coronary vessel. Effectiveness was defined as the percentage of correct diagnosis (cd) and costs were calculated using country-specific reimbursements. Incremental cost-effectiveness ratios (ICERs) were obtained using per-patient data and considering “no-imaging” as reference. The overall prevalence of obstructive CAD was 28%. Strategies combining CTCA followed by stress ECHO, SPECT, PET, or stress CMR followed by CTCA, were all cost-effective. ICERs values indicated cost saving from − 969€/cd for CMR-CTCA to − 1490€/cd for CTCA-PET, − 3092€/cd for CTCA-SPECT and − 3776€/cd for CTCA-ECHO. Similarly when considering early revascularization as effectiveness measure.In patients with suspected stable CAD and low prevalence of disease, combined non-invasive strategies with CTCA and stress-imaging are cost-effective as gatekeepers to ICA and to select candidates for early revascularization.The online version of this article (10.1007/s10198-019-01096-5) contains supplementary material, which is available to authorized users. Currently, available guidelines for symptomatic patients with suspected stable coronary artery disease (CAD) recommend diagnostic flow-charts using computed tomography coronary angiography (CTCA) as the first-line investigation in all patients  or stresDespite this and in view of the wide range of valid available options, the selection of non-invasive imaging in clinical practice may be influenced by factors not included in guidelines . MoreoveWhile innovations in medical imaging have brought unquestionable benefits improving diagnostic and prognostic accuracy of non-invasive pathways, that success is counterbalanced by an escalation of imaging costs. Evidence suggests that the cost of diagnostic imaging has nearly doubled in the past few decades and there is also a wide variation among countries, due to different practice, healthcare and reimbursement systems , 5; thusAccordingly, studies reporting results about cost-effectiveness evaluation of strategies for CAD have slightly increased over the years .In detail, as for clinical studies , availabIn detail, currently available economic studies frequently fail to map all possible options that became available for the diagnosis of stable CAD; many of them use different reference strategies  and indeed most studies focused on the comparison of a single test failing to produce evidence on a combination of different options; contrary to the direction in which the results of the clinical studies are going. As recent reviews of economic studies highlighted, results from available economic evaluations of non-invasive imaging strategy for stable CAD remains controversial, with no modality consistently emerging as superior to all others and across all patient subgroups , 8 and tThe Evaluation of Integrated Cardiac Imaging for the Detection and Characterization of Ischemic Heart Disease (EVINCI) study  was a multicenter, multinational European, non-randomized controlled clinical trial intended to compare the performance of CTCA and available non-invasive stress-imaging modalities for the diagnosis of obstructive CAD in patients with stable chest pain symptoms, intermediate PTP and without prior evidence of disease. The main results of the EVINCI study have been reported elsewhere [The health economic analysis was a major secondary aim of the study, and the EVINCI design allowed comparing strategies involving single modalities or combinations of CTCA and stress imaging for diagnostic cost-effectiveness with the potential of providing valuable evidence and resolving some limitations of available evidence, first and foremost with respect to the evaluation of strategies combining imaging modalities.N = 101) having complete data on ICA, CTCA, and both ECHO and SPECT, the most frequently used imaging stress tests.Per-patient data from the EVINCI study were used in the present analysis. Briefly, the EVINCI trial prospectively enrolled patients with symptoms of suspected stable CAD, without prior evidence of disease and intermediate PTP, after exclusion of those subjects reclassified to very low or high risk after performing an exercise ECG . The stuThirteen strategies involving CTCA and single imaging stress-tests or their combinations were evaluated. Positivity criteria and quality evaluation at core-lab analysis for each non-invasive test were detailed in Neglia et al. . Any tesObstructive CAD was defined as > 50% diameter stenosis in the left main or at least one major coronary vessel by quantitative ICA at core-lab analysis. For all imaging-guided diagnostic strategies true positives (TP) or true negatives (TN) (correct diagnoses) were defined in concordance with ICA results. In patients who did not perform invasive evaluation due to all negative non-invasive exams, and in whom negativity was confirmed by core-labs, all strategies were assumed to be true negatives. For the cost-effectiveness analysis, a “no-imaging” strategy was taken as reference and direct referral to ICA was considered as a comparative pathway. The “no-imaging” strategy was assumed to provide a correct diagnosis only in patients with negative ICA, while the direct invasive strategy was assumed to provide a correct diagnosis in all patients.Effectiveness was defined in terms of correct diagnosis. For each patient and for each strategy, a dichotomous variable of effectiveness was defined taking the value of “one” in case of concordance with ICA results and the value of “zero” in case of discordance.Country-specific charge collected through the EVINCI study was used to quantify costs associated with single tests and combinations. In particular, in each participating center, clinicians and administrative personnel filled a dedicated case report form including code, description, charge and reimbursement regimen for different tests. Collected data were checked against national administrative data, when available, and inconsistencies were solved by direct contacts with the center. Overall costs of the different strategies were thus estimated for each patient adding the specific cost data to costs associated with false negative or false positive results. In published reports –13, the The cost-effectiveness analysis was performed using the payer perspective and the diagnostic performance as measure of effectiveness. Using the “no-imaging” strategy as reference, the cost-effectiveness ratio (ICER) was defined as incremental cost for one more correct diagnosis (€/cd) over 100 patients. Mean costs, effectiveness and the cost-effectiveness ratios (CERs) were derived for all the strategies. Strategies were then ranked based on ascendant order of costs and effectiveness. The ICERs were thus derived as the ratio of incremental costs by incremental unit of effectiveness after excluding strategies “dominated” (less effective and more costly than one or more strategies) or subjected to “extended dominance” (dominated by a linear combination of other strategies). The 95% confidence intervals for ICER were computed by means of non-parametric bootstrap with 10,000 replicates and accounting for the hierarchical structure of the data, with patients clustered into countries.In a scenario analysis, concordance of positive/negative non-invasive strategies results with performed/not-performed early revascularization procedures  was used as an alternative measure of effectiveness.Both the main analysis and the scenario analysis were repeated in the subgroup of the EVINCI population in which each patient had undergone CTCA, stress ECHO, stress SPECT and ICA.The main cost-effectiveness analysis was also performed separately in the different countries.Details of patients’ characteristics, diagnostic performance of single tests and combined non-invasive strategies as well as costs are available as Supplementary data (Tables S1–S3). Briefly, demographics and clinical characteristics of patients undergoing each different imaging were fairly well matched, with patients undergoing PET being less likely males with lower prevalence of family history of CAD and less typical symptoms. The overall prevalence of obstructive CAD was 28% and was similar among groups (Table S1).The diagnostic performance, as expressed by the percentage of correct diagnoses (TP + TN), was in general higher for combined CTCA and stress imaging strategies than for single non-invasive tests (Table S2).Country-specific reimbursement for the different non-invasive tests and ICA largely varied across countries and variability remained substantial also when adjusting for PPP (Table S3).Results of the main cost-effectiveness analysis for the diagnosis of obstructive CAD are summarized in Table The differences in mean cost and in mean effectiveness  are shown in Fig. When considering early revascularization as an alternative measure of effectiveness, combined strategies with CTCA as gatekeeper followed by either stress CMR, ECHO, PET or SPECT were all cost-effective as compared with “no-imaging” and dominated stand-alone or other combined strategies, despite some uncertainty around the point estimate of the ICERs. Direct referral to ICA was cost-effective requiring additional costs Table .Table 2RThe main results of the cost-effectiveness analysis were confirmed in the subgroup of 101 patients submitted to CTCA, stress ECHO, stress SPECT and ICA Table . CombineResults from the country-specific cost-effectiveness analysis indicated high variability among countries and high uncertainty for most of the strategies, mainly due to the limited sample size Figs. , 4 and 5The economic evaluation of the EVINCI study indicates that non-invasive strategies combining CTCA with stress-imaging, in the case of inconclusive results of the first test, are cost-effective diagnostic options in patients with stable chest pain symptoms and low prevalence of obstructive CAD. CTCA as first test followed by stress ECHO, SPECT, PET or stress CMR as first test followed by CTCA were associated with overall reduced costs and higher diagnostic efficacy, as compared with a “no-imaging” strategy, self-standing single non-invasive tests or other combinations. Direct referral to ICA was also a cost-effective option but required additional costs. In general, whether the combination of CTCA with stress-imaging would be used as gate-keeper to catheterization, the positive yield of ICA for the diagnosis of obstructive CAD would increase from 28 to 47% and the rate of revascularizations performed from 19% up to 40% Fig. .Fig. 6ThIn detail, the present study highlights the lower performance of self-standing non-invasive imaging modalities as compared with combined strategies and that could be explained by multiple factors. The EVINCI population, as most recent cohorts enrolled in imaging trials , 18, hadThe present findings cannot be extended to populations with higher prevalence of disease and do not provide information on the impact on long-term clinical outcome. However, from a health economic perspective they support the diagnostic use of integrated non-invasive imaging for the screening of patients with suspected stable CAD recommended in the 2013 ESC Guidelines [Variability in the study design, imaging methods and measures of effectiveness and costs prevents an easy comparison of available studies with the present findings. Many existing studies were performed in selected populations recruited in single or a few expert centers in one nation and did not include all available modalities and combinations. In the CE-MARC study, for example, multi-parametric CMR performed in a single expert center was compared with conventional stress SPECT showing the superiority of strategies including CMR at cost-effectiveness analysis  as also Pragmatic randomized controlled trials (RCTs) are beneficial in that they allow follow-up testing and care patterns to occur based on the patient’s overseeing physician and not constrained by trial-directed follow-up care, thereby reflecting current real-world practices. However, they are also limited by possible inconsistency of patients’ management with imaging results and by variability in the outcome measurements. The PROMISE study was the largest recent pragmatic randomized controlled trial randomizing symptomatic patients without known CAD to CTCA or functional testing including stress SPECT, ECHO or ECG . The oveIn the SCOT-HEART, patients were randomized to standard stress testing, including stress SPECT in a minority, or standard stress testing with a CTCA-guided strategy . There wIn summary, in patients with suspected stable CAD and low-to-intermediate prevalence of obstructive disease, one modality or strategy did not consistently emerge as superior to all others from a health economics perspective , 28.Despite the differences with other cost-effectiveness studies, the EVINCI analysis confirmed that no self-standing non-invasive imaging modality is superior to the others but also showed how strategies combining CTCA with stress-imaging can be cost-effectively used to diagnose obstructive CAD and identify candidates to revascularization prior to ICA.The present study improved the state of the art regarding the cost-effectiveness of strategies for the diagnosis of CAD, first of all by providing evidence in a contemporary European population with acceptable geographical and socio-demographical “representativeness”, both updating evidence about self-standing non-invasive diagnostic strategies and also bringing data for the evaluation of combined strategies.Finally, some other strengths of the EVINCI economic analysis are related to the process of gathering imaging data. These were prospectively obtained in a homogeneous European population of patients with symptoms of suspected stable CAD and low prevalence of disease, and head-to-head comparisons of multiple diagnostic strategies, including combinations, were allowed by the study protocol. Diagnostic effectiveness of tests was also objectively evaluated by independent core-labs and costs assessed in each center.One limitation of the present study regards the diverse utilization of imaging modalities among the single countries and centers. Those differences, despite reflecting real-practice and the different spread of technologies among the involved centers, limited the direct comparison of all the strategies and of modalities exploited at their full potential. Similarly, this is so for the diverse number of patients assigned to the different strategies in different centers and countries. There were insufficient numbers to perform stratified analyses to account for these differences.As the EVINCI study was designed for a diagnostic end-point, correct diagnosis of obstructive CAD was used as measure of effectiveness. This implied the assumption of perfect accuracy of ICA, thus potentially giving CTCA an advantage over stress-imaging. On the other hand, in the scenario analysis based on the revascularizations performed, functional imaging was potentially advantaged.Cost data collected throughout the EVINCI study showed high variability of charges for the different tests among the countries involved (Table S1). Despite this variability, ICA was always the most expensive procedure, while ECHO was the least expensive one, followed by CTCA. SPECT and CMR were generally more expensive than CTCA and associated charges showed the highest variability, even when adjusted for PPP. As previously reported , 28, 29 In a contemporary European population of patients with suspected stable CAD and low prevalence of disease, diagnostic strategies combining CTCA and stress-imaging are cost-effective as gatekeepers to ICA and for selecting candidates for revascularization. In a scenario of uncertainty about health economic consequences of multiple diagnostic pathways, a rational combination of non-invasive imaging modalities may offer the most cost- and clinically effective t!esting for the single patient.The present results suggest the clinical and economic relevance of improving the diagnostic performance of imaging tests and potentially limiting inconclusive imaging findings using more updated technologies, exploiting each tes!t at its full potential  and strictly following correct protocols. They also support the existing recommendations to clearly state when and why an imaging exam is inconclusive in the clinical reports  to indicWhich imaging strategy, beyond its diagnostic performance, is able to achieve the most important goal of effectively guiding treatment to improve outcome in patients with stable CAD is still undetermined. Further large fully comparative multicenter, multinational, multimodality outcome-based imaging trials are needed to provide useful global and country-specific health economic information.Supplementary material 1 Supplementary data detail patients’ characteristics (Table S1), diagnostic performance of single tests and combined non-invasive strategies (Table S2), and centers’ specific charges for the diverse tests (Table S3) (DOCX 74 kb)Below is the link to the electronic supplementary material.


Tuberculosis (TB) infection was responsible for an estimated 1.3 million deaths in 2017. Better diagnostic tools are urgently needed. We sought to determine whether accurate TB antigen detection in blood or urine has the potential to meet the WHO target product profiles for detection of active TB.We developed Electrochemiluminescence (ECL) immunoassays for Lipoarabinomannan (LAM) and ESAT-6 detection with detection limits in the pg/ml range and used them to compare the concentrations of the two antigens in the urine and serum of 81 HIV-negative and -positive individuals with presumptive TB enrolled across diverse geographic sites.LAM and ESAT-6 overall sensitivities in urine were 93% and 65% respectively. LAM and ESAT-6 overall sensitivities in serum were 55% and 46% respectively. Overall specificity was ≥97% in all assays. Sensitivities were higher in HIV-positive compared to HIV-negative patients for both antigens and both sample types, with signals roughly 10-fold higher on average in urine than in serum. The two antigens showed similar concentration ranges within the same sample type and correlated.LAM and ESAT-6 can be detected in the urine and serum of TB patients, regardless of the HIV status and further gains in clinical sensitivity may be achievable through assay and reagent optimization. Accuracy in urine was higher with current methods and has the potential to meet the WHO accuracy target if the findings can be transferred to a point-of-care TB test. Mycobacterium tuberculosis (Mtb), was responsible for an estimated 1.3 million deaths in 2017 [Tuberculosis (TB), an infection caused by  in 2017 . Data sh in 2017 ,6.Mtb antigens in blood or urine provides an attractive alternative approach. Antigen detection is also compatible with the use of simple and low-cost immunoassay techniques, like rapid strip tests, that are better suited to the primary care setting in low and middle income countries. Most work to date on antigen-based assays for TB has focused on detection of lipoarabinomannan (LAM), a Mtb cell wall lipopolysaccharide. It is well established that LAM can be found in the sputum [Diagnosing TB by detection of e sputum –9 and ure sputum –13 of soe sputum –19. Seroe sputum .Commercial implementations of LAM tests have so far been limited to measuring LAM in urine. A commercial lateral flow LAM (LF-LAM) test measuring urinary LAM to diagnose TB is available–the Alere Determine TB LAM Ag test from Abbott Diagnostics–but its adoption has been limited by its poor clinical sensitivity, which a recent meta-analysis estimated at 45% with a 95% confidence range of 29% to 63% . ClinicaMtb. A 2011 review of antigen detection assays for TB lists a number of published reports dating back to the 1990s where research assays for undefined TB proteins were used to test a variety of clinical samples [Mtb, or peptides derived from these proteins, may be found in the serum of TB patients when using new sensitive assay techniques. Specific Mtb proteins that have been measured in serum or plasma include ESAT-6 [Mtb protein have focused on blood-derived matrices. One study provided evidence that ESAT-6 can be found in the urine of TB patients, but this study was limited to urine from four subjects [Relative to LAM, considerably less attention has been given to assays for protein antigens of  samples , but thee ESAT-6 –33, CFP-e ESAT-6 –30,33, Ae ESAT-6 –36, Mpt3e ESAT-6 . Surprissubjects . Anothersubjects . Finallysubjects .In an earlier study , we demoMtb strain Aoyama-B was obtained from Nacalai Tesque Inc. . The Aoyama B strain has been commonly used in Mtb research and has also been used in Japan for tuberculin production [Mtb culture filtrates were provided by the Biodefense and Emerging Infections Research Resources Repository (BEI). The monoclonal antibodies used in the LAM assays have been previously described [Purified LAM from oduction . Recombiescribed ,25,40,42Immunoassays for LAM employing a multiplexed sandwich immunoassay format and ECL detection were carried out on commercial instrumentation and multi-well plate consumables from Meso Scale Diagnostics, LLC. (MSD) . The assThe Immunoassay for ESAT-6 was also conducted using MSD instrumentation and consumables but was carried in a singleplex configuration using MSD’s ultra-sensitive S-PLEX ECL format. The S-PLEX procedure was similar to that described for LAM above, except that instead of using the U-PLEX plates, the capture antibody (Bioporto) was directly immobilized on the carbon ink electrode. In addition, after completion of the detection antibody (MSD) binding step, the S-PLEX format –49 incluMtb culture filtrate (ESAT-6 assay) diluted into a serum-free calibration diluent . Each calibrator concentration was run in duplicate in each assay plate. ESAT-6 concentrations in the Mtb culture filtrate were assigned based on comparison to a recombinant ESAT-6 reference material prepared in E.coli. The relationship of ECL signal to analyte concentration for the calibration curves was fit to a 4-parameter logistic (4-PL) function. Analyte concentrations for test samples were calculated by back-fitting ECL signals to the 4-PL fit and were corrected for sample dilution.Quantitation of the target analytes was based on 8-point calibration curves using purified LAM (LAM assays) or To inactivate any host antibodies in urine and serum samples that could interfere with the assay measurement, the samples were heat-inactivated prior to analysis with the LAM or ESAT-6 assays. Serum samples were diluted 1:4 in PBS prior to heat-inactivation to avoid the formation of clots; urine was heat-inactivated without dilution. For heat-inactivation, both, urine and serum samples were heated to 85°C for 10 minutes or 95°C for 5 minutes.M. tuberculosis complex in cultures was confirmed by Ziehl-Neelsen staining or auramine O fluorescence microscopy to identify acid-fast bacilli, MPT64 antigen detection using rapid speciation assays , or molecular methods. TB-positive individuals were patients with at least one positive culture. All TB-positive patients had positive microscopy results. Participants who were smear negative and culture negative on cultures from all sputum samples and who exhibited symptom resolution in the absence of tuberculosis treatment and negative sputum culture results at a 2-month follow-up visit were classified as TB negative. The subjects were further classified as HIV positive or HIV negative on the basis of HIV rapid tests. More details on sample collection and classification of subjects can be found in the description of our previous urinary LAM study [For this retrospective case-control study, a total of 75 urine samples and 75 serum samples were selected from FIND’s biobank. For all but six of the urine samples, matching serum samples were available that were collected from the same study subjects on the same day. For the remaining six urine samples , serum samples were identified from six additional TB- HIV+ subjects and these were not included in the comparisons that required matched samples. The samples were collected in studies from adults presenting at primary care sites in Bangladesh, Peru, South Africa and Vietnam with clinical symptoms of TB, but not receiving TB treatment at the time of sample collection. Approval by local Ethics Committees and written informed patient consent was obtained before enrolling patients and no personally identifiable information was available to FIND or to the researchers. For use in patient classification, sputum samples  were collected from all participants, decontaminated, and tested in up to six independent liquid cultures  and solid cultures (Lowenstein-Jensen medium). The presence of the AM study . LAM assData analysis was conducted using the R statistical programming language (version 3.5.1). Confidence intervals for proportions were calculated as the Wilson interval using the “binconf” function from the Hmisc package in R. Comparison of groups was carried out by the Mann-Whitney test using the “wilcox.test” function in R. Correlation constants and p values for the null hypothesis of no correlation were calculated using Pearson’s method and the “cor.test” function in R. Correlations of concentration values were determined using log10 transformed concentrations and only included data points where both methods being compared had detectable concentrations. The Cohen’s kappa statistic for categorical agreement was calculated using the “Kappa” function from the vcd package in R. All relevant data are within the manuscript and its Supporting Information files.The analytical sensitivity of the LAM and ESAT-6 ECL immunoassays was established by testing the LAM and ESAT-6 calibration standards diluted in a serum-free calibration buffer. We examined whether optimal assay performance required sample preparation steps to inactivate proteins in urine or serum that could bind the antigen targets and interfere with antibody recognition. In our previous study of LAM in urine, we found no evidence of inhibitory proteins in urine, but we also found no negative impact on measured LAM concentrations if we heated urine samples under conditions that inactivate host antibodies (85°C for 10 min.). In the current study, we investigated the effect of heat inactivation on detection of LAM in serum and ESAT-6 in serum and urine. Mtb cell culture filtrate containing native ESAT-6 was spiked into either urine or serum to provide a final ESAT-6 concentration of 1 ng/mL  was considerably lower than that of the LAM assay using the S4-20 capture antibody . The clinical sensitivity for measuring ESAT-6 in serum  was lower than that observed using urine samples, but was comparable to the sensitivity of the best serum LAM assay using the FIND 28 capture antibody . Good specificity was observed for the ESAT-6 assay in both urine (97%) and serum (100%) when calculated across all subjects regardless of HIV status.We previously reported that the distribution of LAM concentrations in the urine of immunocompetent TB+/HIV+ subjects was not significantly different from the LAM concentrations of TB+/HIV- subjects but that LAM concentrations were higher in immunosuppressed subjects with CD4 cell counts below 100 cells/uL . We exteIn this study, we have shown that LAM and ESAT-6 can be detected in both urine and serum samples of TB patients, using newly developed sensitive ECL immunoassays. The two antigens assayed showed similar concentration ranges within the same sample type. The concentrations were on average roughly 10-fold higher in urine than in serum, but correlated. The measured concentrations of LAM in urine for HIV- patients  were consistent with values reported for this population using a technique involving pre-concentration of LAM from urine using nanocages followed by quantitation by Western blot . It is fThe measured concentrations of ESAT-6 in serum , however, were considerably lower than the concentrations reported by a group using a technique involving trypsin digest of serum samples, concentration of ESAT-6 associated peptides using nanodisks and quantitation by mass spectrometry , who repThe correlation between LAM and ESAT-6 suggest that the two antigens enter urine and blood by similar mechanisms in a large fraction of TB patients. Renal infection has been proposed as a mechanism leading to LAM antigenuria . Renal TMtb specific capture antibody (S4-20) provided the best clinical performance in urine but had relatively poor sensitivity in serum relative to a more generic anti-LAM antibody (FIND 28). The S4-20 antibody targets a unique capping epitope  that is almost exclusively found in TB-causing mycobacteria, while FIND 28 targets branched arabinose structures that can be found in LAM from a variety of mycobacteria and related species [Mtb commensal organisms that can produce LAM and urine collection is more prone to contamination during sampling. We saw no evidence of matrix inhibition of the ESAT-6 assay in either matrix, although the heat inactivation step appeared to improve the signal generated by ESAT-6, even in the absence of urine or serum. While a mechanism for this effect was not determined, potential explanations could be the exposure of epitopes by conformation change (like protein unfolding) on heating or by disruption of protein complexes.Binding of LAM to other antibodies and other proteins in clinical sample matrices can potentially interfere with its detection . We obsedy FIND 2. The S4- species ,40. ThisThe preliminary clinical performance results from our case-control study indicate that the ECL LAM and ESAT-6 assays for urine samples have the potential to meet the WHO sensitivity (>65%) and specificity (>98%) targets for a POC TB diagnostic test, providing support for further research and development of improved POC tests targeting these antigens. LAM in urine remains the most promising target. Although the observed clinical sensitivity for detection of LAM and ESAT-6 detection in serum were lower, blood-based tests could play an important role for TB diagnosis in young children, where urine and sputum collection are less feasible.The clinical performance results presented here provide preliminary evidence for the potential clinical validity of the ECL assays for detecting LAM and ESAT-6 but the generality of the results is limited by the relatively small sample size, the use of a case-control design and the limitation of cases to subjects with smear-positive pulmonary TB. Larger follow-up studies are in progress to better characterize clinical performance through prospective recruitment of relevant and unbiased populations of patients seeking care at TB centers and HIV clinics with symptoms of TB and first results of the Fujifilm SILVAMP TB LAM test are available . It is aOverall, our results suggest that LAM and ESAT-6 are present in urine and serum of TB patients regardless of HIV status. It is difficult to rule out the possibility, particularly for LAM in serum, that there may be forms of LAM present in samples that have epitopes that are either not exposed to or not recognized by our LAM antibodies; further gains in clinical sensitivity may be achievable through assay and/or reagent optimization. The accuracy of blood-based TB tests may also be improved by combining the results of LAM and ESAT-6 measurements, as our results identified subpopulations of TB+ subjects that had detectable concentrations of only one of the antigens. Another option might be the simultaneous detection of the antigen(s) in combination with a host blood marker to come up with a triage test. A recent systematic review indicates that biomarker combinations are more likely to reach the required TPP performance compared to single markers . ResearcS1 TableThe table summarizes the analytical performance of LAM assays using the 6 different anti-LAM capture antibodies, as well as the performance of the ESAT-6 assay. The results were determined from 8 point calibration curves as described in (DOCX)Click here for additional data file.S2 Table(a) Effect of heat inactivation on spike recovery. LAM was spiked into three normal urine samples (Neg Urine), three normal serum samples (Neg Serum) or a simple buffer (Diluent). The concentrations of LAM were measured in each of these samples with each of two LAM capture antibodies (FIND 28 or S4-20), with (Heat) or without (No Heat) pre-treatment of the spiked sample by heat inactivation. The table provides the concentrations normalized to the measured level in diluent without pre-treatment (% Recovery). (b) Effect of heat inactivation on assay signals for samples from TB+ individuals. LAM was measured in three urine samples and three serum samples from TB+ individuals (Pos Urine and Pos Serum). The table provides the assay signals with and without pre-treating the samples with heat inactivation, and also provides the fold-increase in signal with pretreatment (Ratio).(DOCX)Click here for additional data file.S3 Table(a) Effect of heat inactivation on spike recovery. ESAT-6 (~ 1 pg.mL) was spiked into three normal urine samples (Neg Urine), three normal serum samples (Neg Serum) or a simple buffer (Diluent). The concentrations of ESAT-6 were measured in each of these samples with (Heat) or without (No Heat) pre-treatment of the spiked sample by heat inactivation. The table provides the concentrations normalized to the measured level in diluent without pre-treatment (% Recovery). (b) Effect of heat inactivation on assay signals for samples from TB+ individuals. ESAT-6 was measured in three urine samples and two serum samples from TB+ individuals (Pos Urine and Pos Serum). The table provides the assay signals with and without pre-treating the samples with heat inactivation, and also provides the fold-increase in signal with pretreatment (Ratio). Note that the samples used to generate this data are not the same samples used to generate the data in (DOCX)Click here for additional data file.S4 TableResults are shown for (a) LAM measurements using the FIND 28 capture antibody, (b) LAM measurements using the S4-20 capture antibody, and (c) ESAT-6 measurements. Below each table are point estimates and 95% confidence intervals for the categorical agreement and Cohen’s kappa statistic.(DOCX)Click here for additional data file.S5 TableBelow each table are point estimates and 95% confidence intervals for the categorical agreement and Cohen’s kappa statistic.(DOCX)Click here for additional data file.