Adaptation to pollution has been studied since the first observations of heavy metal tolerance in plants decades ago. To document micro‐evolutionary responses to pollution, researchers have used phenotypic, molecular genetics, and demographic approaches. We reviewed 258 articles and evaluated the evidence for adaptive responses following exposure to a wide range of pollutants, across multiple taxonomic groups. We also conducted a meta‐analysis to calculate the magnitude of phenotypic change in invertebrates in response to metal pollution. The majority of studies that reported differences in responses to pollution were focused on phenotypic responses at the individual level. Most of the studies that used demographic assays in their investigations found that negative effects induced by pollution often worsened over multiple generations. Our meta‐analysis did not reveal a significant relationship between metal pollution intensity and changes in the traits studied, and this was probably due to differences in coping responses among different species, the broad array of abiotic and biotic factors, and the weak statistical power of the analysis. We found it difficult to make broad statements about how likely or how common adaptation is in the presence of environmental contamination. Ecological and evolutionary responses to contamination are complex, and difficult to interpret in the context of taxonomic, and methodological biases, and the inconsistent set of approaches that have been used to study adaptation to pollution in the laboratory and in the field. This review emphasizes the need for: (a) long‐term monitoring programs on exposed populations that link demography to phenotypic, genetic, and selection assays; (b) the use of standardized protocols across studies especially for similar taxa. Approaches that combine field and laboratory studies offer the greatest opportunity to reveal the complex eco‐evolutionary feedback that can occur under selection imposed by pollution. HoweverWabs) results in population decline  and resulting in population recovery through the process of evolutionary rescue  by means of plastic responses without changes in genetic composition  identify a trait(s) that can provide a fitness advantage in dealing with the stressor, (b) assess the presence of suitable genetic variation for the particular trait(s); (c) show that selection (as opposed to genetic drift) has taken place; (d) assess the contribution of the advantageous trait(s) to the population fitness by estimating population growth rate Bell, . AlthougSeveral literature reviews have summarized studies focused on the ability of organisms to tolerate pollutants , taxonomic groups , methods (field and laboratory studies), and pollutants . We performed a quantitative meta‐analysis with a subset of the data to evaluate the effect of metal pollution on the magnitude of phenotypic response  in invertebrates. We also evaluated how shifts in methodological approaches have changed our understanding of micro‐evolutionary responses to pollution. In particular, we assessed molecular evidence for adaptation and the candidate genes potentially involved in the pollution‐induced evolutionary processes .2Google Scholar. We used the keywords “genetic adaptation,” “adaptation,” “micro‐evolution” in combination with “pollution” or “pollutants” or “contaminants.” These search terms reduced bias to a particular approach or method.In this study, we reviewed articles published since 1992 found by searching on 2 increase), polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and various other chemical pollutants . In these cases, random genetic drift was ruled out. Since it is common in evolution studies to substitute time for space and use geographic variation in resistance as an alternative for temporal changes /3.92. When the sample size was <60, we replaced 3.92 with values obtained from tables of the t distribution with degree of freedom equal to the group sample size minus 1.We identified a set of response variables that are measured in laboratory and field experiments to study the evolution of resistance through molecular markers and/or quantitative traits. Studies were then assessed and classified according to the approach(es) used: (a) analysis of phenotypic responses to pollution leading to resistance (“phenotypic assays”); (b) characterization of the genetic basis/underlying genetic variation of the advantageous phenotypic traits (“genetic assays”); (c) tests for evidence of selection against random genetic drift and gene flow (“selection assays”); (d) assessment of population growth rate  to estimate the displayed values. When sample size was given as a range, we calculated the average. The dataset included a large variety of experimental designs that were mainly classified as either “field” or “experiment”  and created a numerical metric for the analyses: the natural logarithm of the metal concentration divided by the threshold concentration specific for each metal and habitat type determined by the Canadian Council of Ministers of the Environment (Supporting Information Table ρ) to calculate the variance components for the between‐study and within‐study heterogeneity. We also recorded the number of generations studied in the subset of papers used for the meta‐analysis (n = 108). Finally, we mapped all study sites to show where pollutants have been studied around the world. For the meta‐analysis, we used the R packages metaphor  focused on metal pollution  were on invertebrates, followed by vertebrates (n = 74), plants (n = 60) and algae (n = 17). Specifically, metals were the most studied pollutants for terrestrial arthropods and to a lesser extent for terrestrial annelids, aquatic arthropods, and mollusks. Plants were also studied primarily in relation to metals. The effect of acidification on algae, invertebrates (Echinodermata), and vertebrates was investigated in 8% of the studies. Polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) were the least represented (9%) with a dozen studies almost exclusively conducted on fish. The rest of the studies (~20%) focused on pollutants like pesticides, dioxin‐like compounds, radiation, waste heap, tributyltin, and other chemical compounds and were grouped together as “other” , Chironomus (invertebrate: Insect), Daphnia (invertebrate: Branchiopoda), Arabidopsis (plant: Eudicots), Orchesella (invertebrate: Entognatha) was common. These species were so extensively studied that they have been the subject of several important literature reviews. For example, Weis  followed by studies on trait heritability (n = 16). Selection assays were the second most abundant assays (n = 121) after phenotypic assays. Studies on invertebrates were the most common followed by vertebrates and plants. FST‐based outlier tests were the dominant way to find evidence for selection. Together with survival, these tests were the most commonly used to study adaptive responses of organisms in polluted conditions. Among the studies that found evidence of selection, the majority (86.5%) tested also for a link between responses to selection and the environment: 15.7% found evidence for genotype–environment correlations and another 15.7% for phenotype–environment correlations (Supporting Information Table n = 31) on invertebrates and microalgae included population growth rate estimates.Phenotypic assays included the study of survival in different concentrations of pollutants , changes in development , growth , morphology , physiology , and reproductive traits . Survival and reproductive traits were the most studied in invertebrates, while in plants and vertebrates all traits were more or less equally studied Figure . Genetic3.2After grouping the results from different articles, we obtained 198 studies Figure , 63% of n = 47) followed by physiological traits (n = 26) and morphological traits . They found that populations from contaminated soil and some populations sampled downstream of the mine had 100% tolerance while populations sampled upstream showed variable tolerance that was related more to geographic location than copper concentration in the soil. Through the study of life‐history traits during experimental selection, they were able to demonstrate that tolerance was heritable and widespread in populations from contaminated soil due to beneficial genetic variation. Shirley and Sibly  was also affected by pollution. Moreover, Messiaen et al. , mathematical models can be crucial for strong inferences about the long‐term effects of pollution on fitness.Kelly et al.’s  study ony Figure . The eff4We tested the relationship between metal concentration and effect sizes of weight, number of neonates, and body metal content. We accounted for the phylum and subclass of the studied organism, the habitat, the type of metal, and other factors such as the presence of other metals in the original habitat and the study type (laboratory experiment or field monitoring). We expected that body weight and number of neonates would decrease with increasing metal concentration and that body metal content would increase.SE = 0.16; Supporting Information Figure ρ) were quite large for body weight  demonstration that the changes in the trait studied are genetically determined and are subject to natural selection; (b) assessment of potential confounding environmental variables; (c) the demonstration that the increase in adaptive trait value can sustain a positive population growth rate and thus the long‐term persistence of the population  long‐term monitoring programs of populations in polluted habitats that integrate demographic studies with phenotypic, genetic, and selection assays; (b) use of standardized protocols among studies of similar species to make evolutionary toxicology studies more comparable (Oziolor et al., Data for this study will be available at Fig Share: 10.6084/m9.figshare.7730255.None declared. Click here for additional data file.


IGF) axis plays key roles in normal tissue growth and development as well as in the progression of several tumour types and their subsequent growth and progression to a metastatic phenotype. This review explores the role of IGF system in normal germ cell development and function in addition to examining the evidence for deregulation of IGF signalling in cancer, with particular relevance to evidence supporting a role in testicular germ cell tumours (TGCTs). Despite the clear preclinical rationale for targeting the IGF axis in cancer, there has been a lack of progress in identifying which patients may benefit from such therapy. Future employment of agents targeting the IGF pathway is expected to concentrate on their use in combination with other treatments to prevent resistance and exploit their potential as chemo‐ and radiosensitizers.The insulin‐like growth factor ( INSR gene resulting in two IR subunits IR‐A and IR‐B, which have differing affinities for insulin and the IGF ligands  signalling system has been implicated in a vast array of both physiological and pathological cellular processes. The IGF family principally comprises three ligands , three receptors  in addition to six high affinity ligand binding proteins (IGFBP1‐6), and accessory proteins such as IGFBP‐specific proteases. The signalling through IR/IGFR is complex , resulting in full recovery of testicular weight and reversal of all histopathological abnormalities  led to infertility  leads to defects in migration of these cells to the genital ridge  and decreased serum levels of some IGFBPs (associated with suppressing growth through binding IGF1) with additional cancer risk  correlate with higher cancer risk and with increased circulating IGF1 levels and/or decreased IGFBP levels Nunney, . Consistet al., increased IGFBP3 levels were associated with risk of pre‐menopausal breast cancer. This finding challenges the assumption that IGFBP3 only exerts its effects on cancer risk by regulating bioavailability of IGF1. This study did not detect a protective effect of lower IGFBP3 levels overall; however, when one of the lung cancer cohorts was removed (that recruited only heavy smokers and asbestos workers), the risk of lung cancer was significantly decreased in individuals with higher IGFBP3 concentration /Igf1 serum concentration had a higher incidence and reduced latency of mammary tumours but only in the context of a high fat diet  pathways. Whereas activation of the MAPK pathway drives cellular proliferation through promoting proteins involved in cell cycle progression, signalling via the AKT pathway both activates anti‐apoptotic proteins and inhibits anti‐apoptotic proteins to enhance cell survival  and tumour‐associated endothelial cells (TECs) may co‐operate in IGF‐mediated chemoresistance. TAMs and myofibroblasts were found to be the main sources of IGF production in pancreatic cancer  and small molecule tyrosine kinase inhibitors of IGF1R (TKIs). These inhibitors to IGF1R were very enthusiastically explored in many clinical trials as single agents two decades ago. Outcomes in these trials were extremely disappointing due to an overall infrequency of objective responses and a lack of any accurate predictive biomarkers of response. The most promising patient subgroup who might benefit from IGF1R‐targeted therapy are Ewing's sarcoma patients, where a minority have sustained durable responses lasting several years without major side effects , identifies patients that would benefit from IGF1R‐targeted agents (Cao et al., et al., PIK3CA or AKT1 mutations are driving resistance, inhibition of the IGF signalling pathway should take place downstream of these. The molecular pathways that allow IGF1R inhibitors to act as chemo‐ or radiosensitizers are not yet fully understood. Identifying these mechanisms and studying their interaction with the deficiencies in DNA repair in TGCT cells will be necessary in order to exploit the full benefit of targeting the IGF axis. The mTOR inhibitor, everolimus, has shown limited efficacy in two phase‐II studies of unselected TGCT patients with refractory disease (Mego et al., et al., In order to exploit the anti‐tumourigenic responses to INSR/IGF1R inhibition seen in preclinical experiments, current investigations are concentrating on combinatorial studies using IGF inhibitors. Combination with other RTK TKIs is used as a means of reducing the emergence of resistance or in addition to either standard chemotherapeutic agents or radiotherapy utilizing their properties as chemo‐ and radiosensitizers, respectively (McDermott et al., et al., Primordial germ cells, the likely precursor of TGCT, require IGF1R signalling for correct migration to the genital ridge, and the IGF system has many roles in establishing and maintaining testicular function including steroidogenesis and maintaining pluripotency in spermatogonial stem cells. The IGF axis is dysregulated in many tumour types and can contribute to oncogenesis via multiple disparate mechanisms, making it an attractive therapeutic target. The lack of mutations found in IGF proteins in cancer may hint that INSR/IGF1R signalling is not a key driver in many tumours, and together with cross talk between pathways, this could explain the lack of efficacy seen in clinical trials using several different types of IGF1R‐targeted agent. However, there are multiple lines of evidence to suggest that cancers can use the INSR/IGF1R pathway as a resistance mechanism to other treatments and that IGF1R inhibition can augment responses to standard chemo‐ and radiotherapy. The clinical utility of blocking this pathway may therefore lie in combining newly designed IGF ligand‐targeted therapies with existing or new treatments. TGCT cells commonly exhibit aberrant IGF axis activation through elevated IGF1R activity (Selfe


A major controversy in psychiatric genetics is whether nonadditive genetic interaction effects contribute to the risk of highly polygenic disorders. We applied a support vector machines (SVMs) approach, which is capable of building linear and nonlinear models using kernel methods, to classify cases from controls in a large schizophrenia case–control sample of 11,853 subjects  and compared its prediction accuracy with the polygenic risk score (PRS) approach. We also investigated whether SVMs are a suitable approach to detecting nonlinear genetic effects, that is, interactions. We found that PRS provided more accurate case/control classification than either linear or nonlinear SVMs, and give a tentative explanation why PRS outperforms both multivariate regression and linear kernel SVMs. In addition, we observe that nonlinear kernel SVMs showed higher classification accuracy than linear SVMs when a large number of SNPs are entered into the model. We conclude that SVMs are a potential tool for assessing the presence of interactions, prior to searching for them explicitly. The assumption of additivity has been an extremely effective approach, but it is also pragmatic, since looking at the effects of many 100,000 s of single nucleotide polymorphisms (SNPs) would be rendered computationally expensive if all potential combinations of interactions were considered Cordell, . In addiThe extent to which genetic interactions contribute to risk is unknown. One study, which explicitly modeled and tested gene expression traits for evidence of pairwise SNP interaction effects  and (b) SNPs with suggestive evidence for an association with schizophrenia (p ≤ .01), was AUC = 0.58 and 0.70, respectively. In the present study, we employ SVM algorithms which offer both linear and nonlinear modeling options and hence may account for pairwise and higher order SNP interactions, to explore a large schizophrenia case–control sample of 11,853 subjects  for classification of SZ cases and controls. We use GWS SNPs and SNPs with suggestive evidence for association with schizophrenia.A landmark GWAS of schizophrenia, conducted by the Schizophrenia Working group of the Psychiatric Genomics Consortium , identified 128 genome‐wide significant SNPs , representing 108 independent loci  region (chr6: 25–34 Mb) was removed due to the highly correlated nature of the SNPs in this region. The imputed data were converted into the most probable genotypes (probability >0.9), and Linkage Disequilibrium (LD) pruned, keeping the schizophrenia associated SNPs and removing those in LD . For SNP prioritization purposes, we used genome‐wide association summary statistic data, (available at https://www.med.unc.edu/pgc/results-and-downloads) of the PGC2 study  and tested those for presence of potential hidden interactions. The choice of this number of SNPs  was dictated by the sample size, to avoid the problem of overfitting the data when the number of features (SNPs) exceeds the number of individuals  in the SVM analyses, for which pairwise interactions were statistically tested (and not found) elsewhere  for each model were selected by randomly sampling from exponential probability distributions C ~ Expon(λ = 1) for both kernels, and, additionally, γ ~ Expon(λ = 0.01) for the RBF kernel. These exponential probability distributions were used since our preliminary analysis trials suggested that lower values of the hyper‐parameters tended to result in higher classification accuracy. Each selection of hyper‐parameters was assessed by taking the mean score across the four folds of the training data. The near optimal hyper‐parameters C = 1 for the SVM‐Linear model and C = 0.5 and γ = 0.02 for the RBF kernel SVM model were selected and used for these 100 simulations below.The data analyses were carried out in Python, using two main packages (scikit‐learn and pandas) for machine learning and data processing  of such splits for each of the different kernels to obtain distributions of accuracy scores for each model. The metric used for all of the performance assessment was the Area Under the receiver operating characteristic (ROC) curve (AUC) metric, also known (ROC) score Metz, .2.3p‐value) are sufficient as this dataset is used to select the SNPs, the risk score alleles and their genetic effects. The second dataset is used generate the PRS for each individual and requires individual genotypes. The PRS for each subject is calculated as a sum of risk alleles in the second dataset, weighted by the SNP effect sizes (β‐coefficients) derived from the first dataset. Logistic regression analysis is then used to assess whether the PRS distributions are different in cases and healthy individuals and to estimate the classification accuracy. This method is widely used in medical and population genetics  from the 75% of the data. As above, this procedure was repeated 100 times.PRS is a method to summarize the trait variance captured by a set of genetic variants. We followed the approach previously described by the International Schizophrenia Consortium  in each data set (Batch 1 and Batch 2) separately, and in the combined dataset (Batch 1 + Batch 2).Finally, the performances of the PRS and SVM models were compared using a 3@@Overall, the maximal prediction accuracy achieved by SVM was AUC = 0.60–0.66, indicating that this approach did not give a practically applicable model for prediction of schizophrenia using these data. The following results focus on the comparison of the performance of the linear and nonlinear prediction modeling.3.1Initially, we built and tested models using the 125 GWS SNPs in the Batch 1 dataset only .N = 100).The linear kernel SVM showed similar prediction accuracy values (AUC‐ROC scores) across different values of C. Figure The results Figure  show thap = .895 and p = .2, respectively), whereas in contrast, the accuracy of SVM‐Linear model did improve (p = .005). However, the fact that the accuracy of the RBF kernel SVM did not improve with the larger sample indicates that it is unlikely that there are detectable nonlinear effects in the data . The latter result replicates the finding of an absence of interactions between these SNPs from , but the PRS model did not (p = .525), see Figure Following a similar strategy to the above, we compared the analyses on the data for 4,998 independent top SNPs, initially in the larger Batch 1 data and then in the combined dataset (Batch 1 + Batch 2). The accuracy of PRS and SVM‐RBF models improved compared to models built upon the 125 GWS SNPs  obtained with multivariate logistic regression (model including each SNP as an independent variable) and PRS for two independent SNPs with effect sizes OR = 1.2 and MAF = 0.3 in 1,000 simulations. The p‐values for PRS analyses are systematically lower than for multivariate regression. The effect is even more pronounced when the number of SNPs increases. We also compared the prediction accuracy of the linear kernel SVM and multivariate logistic regression models. The accuracy, as expected, was very similar (results are not shown). Since both the multivariate regression and SVM‐Linear models include all SNPs as separate variables for prediction, a direct comparison of the SVM‐Linear model with the multivariate regression analysis is natural. However, such comparison was not viable in our schizophrenia samples due to the limitation on the number of predictor variables in multiple linear regression modeling for accurate estimation of regression coefficients  SVM models did not improve the prediction accuracy over the SVM‐Linear model. This implies that it is unlikely that there are SNP × SNP interactions among this set of SNPs with sufficiently large interaction effect sizes to be detected with the current sample size. When expanding analyses to a larger set of SNPs, for the Batch 1 dataset the performance of the linear kernel decreased, while that of the RBF kernel showed a modest increase. The addition of the data samples from Batch 2 resulted in a clear increase in prediction accuracy in both models. This increased benefit of including the additional samples was not seen in the models using only the GWS SNPs. In addition, we observed that the SVM model with RBF kernel displayed superior performance over the linear kernel, indicating possible evidence for interactions between the SNPs. In contrast, the prediction accuracy of PRS did not increase with the sample size. To corroborate this evidence, it would be informative to explore whether the SNPs contributing most to SVM classification tend to contain interaction effects; however, current nonlinear SVM implementations do not naturally provide information on which SNPs these are.Neither of the SVM models demonstrated a better predictive accuracy than the polygenic risk score approach. However, we have shown that due to the selection of SNPs for the PRS, the PRS approach will have an advantage in terms of statistical power and prediction accuracy over multivariate analyses, including SVM with linear kernel and multivariate regression analyses. We note that the PRS score can be adjusted to remove the SNP–SNP correlation in the data, which makes the PRS approach essentially equivalent to multivariate regression (Yang et al., In conclusion, the RBF kernel prediction accuracy shows substantial improvements over the results from the linear kernel in real data with 4,998 SNPs. This finding indicates the potential presence of interactions between these relatively weakly associated schizophrenia genetic variants, over and above the main effects of these SNPs, whilst we found no evidence for interactions among genome‐wide significant index SNPs. Based on the results of the present study, we conclude that PRS remains the better option for the purpose of classification of schizophrenia cases from controls, although its currently demonstrated accuracy is insufficient for clinical practice.The authors have no conflict of interest to declare.


Self‐reported influenza vaccination is generally accurate for the current season, but the accuracy of self‐report for vaccination in prior seasons is largely unknown. This study evaluated the accuracy of self‐report for current and prior season influenza vaccination among patients with medically attended acute respiratory illness enrolled in a study of influenza vaccine effectiveness during the 2014‐15 influenza season. It demonstrates there is a greater potential for exposure misclassification when prior season vaccinations are ascertained by self‐report. Percent agreement between self‐report and final status was high for both current and prior season vaccination: 97.7% and 93.2%, respectively. Accurate information on influenza vaccination status is important to minimize exposure misclassification and bias. These studies often rely on a combination of self‐report and information obtained from medical records to determine vaccination status. In the United States, influenza vaccines are readily available outside the healthcare system, and self‐report may be the only practical method to ascertain vaccination status in some research settings. This is especially true for ascertainment of vaccines received in prior seasons. Vaccine effectiveness may be reduced in persons who are repeatedly vaccinated, and recent VE studies have assessed this by collecting information on prior season vaccination. Self‐report of influenza vaccination is generally accurate for the current season, but the accuracy of self‐report for vaccination in prior seasons is largely unknown.2This study was conducted within an influenza vaccine effectiveness study in the 2014‐15 northern hemisphere influenza season. The methods of the influenza vaccine effectiveness study have been previously reported.Vaccination status was ascertained from a regional immunization registry. The Registry for Effectively Communicating Immunization Needs (RECIN) is a web‐based, population‐based immunization registry to capture vaccines for both adults and children. RECIN is utilized by all public and private immunization providers serving the study population in central Wisconsin. The registry contains a longitudinal record of immunizations received by each individual, exchanges data weekly with the Wisconsin Immunization Registry, and employs record‐matching procedures to prevent duplicate entries. RECIN data include vaccine type, manufacturer, lot number, date of administration, and name/location of immunization provider. RECIN is integrated into the Marshfield Clinic electronic medical record and serves as the legal immunization record.For this analysis, the accuracy of self‐reported vaccination was assessed relative to RECIN records at the conclusion of the season. If a RECIN record indicated receipt of influenza vaccine prior to enrollment in 2014‐15 or at any time during the 2013‐14 season, the patient was classified as vaccinated for the respective season. Records of patients who self‐reported no vaccination for which RECIN contained an influenza vaccination record were reviewed to confirm identity matching. If matching was confirmed, the RECIN record was accepted as valid. Participants who reported influenza vaccination in 2013‐14 or 2014‐15 without a corresponding RECIN record were interviewed by telephone. Those who re‐confirmed that they (or their child) received an influenza vaccine in the season(s) under investigation were asked to provide the date, location, and provider who administered the vaccine. Written documentation of vaccination was requested. Following consent for release of medical records, immunization providers were contacted to confirm vaccine receipt. For participants reporting receipt of influenza vaccine from Marshfield Clinic, health system electronic medical records were reviewed to confirm vaccination.Participants were classified as vaccinated if (a) a record existed in RECIN or (b) written documentation of vaccine receipt was obtained from the immunization provider, or (c) health system electronic medical records confirmed vaccination. Participants were classified as unvaccinated if (a) there was no record of vaccine administration in RECIN and (b) confirmation of no vaccination was received from an immunization provider or health system electronic medical records had no evidence of vaccination.We calculated percent agreement and kappa between self‐reported and adjudicated vaccination status. The sensitivity was defined as the percent of vaccinated individuals correctly identified as vaccinated by self‐report. Specificity was defined as the percent of unvaccinated individuals correctly identified as unvaccinated by self‐report. Participants were excluded if responding “don't know” to vaccination status, refusing participation, unable to be reached, unable to provide sufficient detail for contacting the immunization provider, or if, after 3 attempts, authorization releases or verification documents were not returned. Children under 6 months of age as of September 1, 2013 were excluded from the prior season analysis as they were not eligible for vaccination. All analyses were performed with SAS 9.4.The study was approved by the Marshfield Clinic Institutional Review Board, and informed consent was obtained from all participants.3The analysis included 1841 patients with acute respiratory illness who were enrolled in the 2014‐15 vaccine effectiveness study; 1683 (91%) were non‐Hispanic white, 89 (5%) were Hispanic, 1025 (56%) were female, and 994 (54%) were aged <18 years. Nine hundred eighty‐four participants (53%) initially reported receiving the 2014‐15 influenza vaccine, and 922 (94%) of these were confirmed by a vaccination record RECIN. There were 62 participants who reported vaccine receipt without a corresponding record in RECIN  (Table For the prior 2013‐14) season  (Table 4This study confirms previous, published reports indicating that self‐report of influenza vaccination in the current season can provide a valid measure of vaccine exposure when medical records or registry data are not available.The lowest percent agreement for report of prior season vaccination status was seen in 18‐ to 49‐year‐olds. Misclassification occurred in nearly 10% of reports in this population. However, for the current season, the percent agreement for the same age group was among the highest, with no one incorrectly reported as unvaccinated and only 2% incorrectly reported as vaccinated without substantiation. This age group had the lowest influenza vaccination coverage in the United States in both 2013‐14 and 2014‐15 and was the last age group recommended for annual influenza vaccination.Adults aged 50 years and over had the highest agreement in both the current and prior season. These findings are concordant with studies showing high reliability of and high sensitivities for self‐report of influenza vaccinations in older adults.In both seasons, the number of vaccinated individuals denying vaccination was less than those reporting vaccination without documentation. There was no observable pattern of late‐season enrollments coupled with early season vaccination which might suggest recall bias within a season, but this misclassification did occur more for prior season vaccination than current season.A limitation of this study is the lack of racial/ethnic diversity. Results may not be generalizable to more diverse urban populations. We were also unable to obtain responses from all vaccination providers, particularly companies hired by employers to implement workplace vaccination campaigns, which led to exclusions from the analyses.In conclusion, this study demonstrates a greater potential for exposure misclassification when prior season vaccinations are ascertained by self‐report. The impact of this exposure misclassification on vaccine effectiveness estimates requires further assessment.


Exosomes are abundantly secreted extracellular vesicles that accumulate in the circulation and are of great interest for disease diagnosis and evaluation since their contents reflects the phenotype of their cell of origin. Tumor‐derived exosomes (TDEs) are of particular interest for cancer diagnosis and therapy, since most tumor demonstrate highly elevated exosome secretion rates and provide specific information about the genotype of a tumor and its response to treatment. TDEs also contain regulatory factors that can alter the phenotypes of local and distant tissue sites and alter immune cell functions to promote tumor progression. The abundance, information content, regulatory potential, in vivo half‐life, and physical durability of exosomes suggest that TDEs may represent a superior source of diagnostic biomarkers and treatment targets than other materials currently under investigation. This review will summarize current information on mechanisms that may differentially regulate TDE biogenesis, TDE effects on the immune system that promote tumor survival, growth, and metastasis, and new approaches understudy to counteract or utilize TDE properties in cancer therapies. Cancer cells exhibit greater exosome secretion rates than nonmalignant cells of the same cell type,TDEs, however, are also known to exhibit significant negative regulatory effects on the immune system that can promote immune tolerance and tumor escape from immune surveillance and there has been considerable research focused on this topic. This review will therefore focus on mechanisms that may regulate enhanced exosome production, how these are altered in tumor cells, regulatory effects of TDEs on the immune system, and the status of approaches to directly or indirectly utilize TDEs in new cancer therapeutics that are currently in development.1.1Exosome secretion requires three distinct processes: the accumulation of ILVs to produce MVB, the migration of these ILV‐rich MVBs to the plasma membrane (or to lysosomes for degradation), and the fusion of MVBs with the plasma membrane to allow the release of their contents into the extracellular space as mature exosomes Figure . Exosome1.1.1Exosomes demonstrate enrichment of the sphingolipids sphingomyelin and hexosylceramide relative to the lipid content of other cellular membranes, and the ceramide content of the outer endosome membrane appears to play an important role in exosome biogenesis. Neutral sphingomyelinase activity can hydrolyze sphingomyelin to ceramide to promote the inward curvature of the outer leaflet of the endosome membrane into the intraluminal space of the endosome to initiate or promote ILV formation.Multiple studies have reported alterations in sphingolipid metabolism in different cancer types, but it is not clear there is a consistent shift toward increased ceramide accumulation in these cancers or that the observed changes promote phenotypes that would be expected to enhance exosome biogenesis.ILVs are known to form through both ESCRT‐dependent and ESCRT‐independent mechanisms Figure , althougThe ESCRT‐0 complex initiates the first step of the ILV biogenesis process by binding and promoting the aggregation of ubiquitinated proteins and regulating the localization of these proteins, and other ESCRT complexes, to clathrin‐coated microdomains on the endosome membrane. The ESCRT‐0 complex contains two subunits, HRS and STAM, both of which contain ubiquitin‐ and clathrin‐binding domains, while HRS also contains a Fab 1, YOTB, Vac 1, and EEA1 (FYVE) zinc finger domain that binds to phosphatidylinositol 3‐phosphate to recruit the ESCRT‐0 complex to the endosome membrane.It is not clear if cancer cell changes or tumor microenvironment stimuli can directly enhance the expression or activity of ESCRT‐dependent or ‐independent MVB biogenesis mechanisms, but the ESCRT‐0 protein HRS and the ESCRT‐I have been reported to demonstrate enhanced expressed in some tumor tissues, suggesting that such mechanisms may exist.1.1.2Mature MVBs can experience different fates depending on cellular conditions at the time of their formation, either trafficking to the cell periphery to permit a subsequent regulated fusion event with the plasma membrane that releases their cargoes of exosomes into the extracellular space or migrating to and fusing with lysosomes or autophagosomes to allow the degradation of their contents. The conditions, processes and factors that control the targeting of MVBs to each of these fates is not completely understood, although one group has now reported that interferon‐stimulated gene 15 (ISG 15)‐mediated ISGylation appears to play a key role in targeting mature MVBs to lysosomes or autophagosomes.Endosomes and mature MVBs are transported along microtubules by dynein/dynactin or kinesin motor complexes in a process regulated the activity of RAB family GTPases, with the inward movement of maturing endosomes regulated by the action of RAB5 and outward transport of mature MVBs regulated by RAB7A.1.1.32+ regulated MVB fusion with the plasma membrane is reported to involve interaction between plasma membrane‐associated SNAP‐23 and MVB‐associated vesicle associated membrane protein (VAMP7), VAMP8, or Syntexin‐4, depending upon the cell type.Exosome release from MVBs docked at the plasma membrane appears to be regulated by GTPase‐mediated interactions between soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) proteins present on the MVB and plasma membranes. Multiple SNARE proteins have been implicated in this process. For example, Ca1.2Most tumors demonstrate an enhanced degree of exosome secretion and several characteristics commonly associated with tumor microenvironments can regulatory exosome secretion rates. For example, most cancers demonstrate a characteristic metabolic shift toward aerobic glycolysis so that pyruvate produced during the first stage of aerobic glucose metabolism is converted to lactate, rather than entering the tricarboxylic acid cycle and undergoing complete oxidative phosphorylation.1.2.1Hypoxia can increase EV secretion rates and while the mechanism responsible is for such increases is not well understood, this effect appears to be hypoxia‐inducible factor 1α (HIF‐1α)‐dependent, since cells treated with HIF‐1α short hairpin RNA or specific‐inhibitors or with HIF‐1α deletions exhibit attenuated EV secretion when incubated under hypoxic conditions.1.2.22 production by the pentose phosphate pathway.Local acidification is a common feature of most tumors since acidic metabolites can accumulate due both to altered tumor metabolism and a relative reduction in the perfusion rate of the tumor. Lactic acid accumulation appears to be a significant contributor to this process, but other mechanisms have also been reported to influence tumor pH, including increased COSeveral lines of evidence indicate that tumor acidification may play important roles in regulating the secretion and function of TDEs. For example, exposure to low pH is reported to stimulate exosome release and uptake through a proposed pH‐dependent effect on exosomal lipid composition,1.2.3Dysregulated growth factor signaling, due to increased expression or altered receptor signaling pathway responses, is associated with enhanced tumor development and progression. Multiple growth factor signaling pathways now know to contribute to distinct tumorigenic responses in different cancer types. However, tumor‐associated epidermal growth factor receptor (EGFR) signaling is particularly notable in that enhanced expression of EGFR or its ligands is detected in most human carcinomas, EGFR expression is induced by local tissue hypoxia, and stimulation of EGFR signaling is reported to enhance exosome secretion.1.2.4Chronic inflammation is reported to regulate all stages of tumor development, from tumor initiation to metastasis,Multiple studies have examined TDE effects on tumor development and tumor microenvironment inflammation, as described in the following sections, but relatively few have examined how stress and proinflammatory tumor cell responses influence exosome secretion. However, recent work suggests that increased NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome activity, which plays a key role in innate immunity, can regulate EV secretion, with several reports demonstrating direct associations between NLRP3 inflammasome activation and enhanced exosome release rates and changes in exosome cargo compositions.The NLRP3 inflammasome is a multi‐subunit cytosolic protein complexes that assembles upon exposure of its pattern recognition receptor, NLRP3, to a broad range of damage‐associated molecular patterns or responses induced by them, including extracellular adenosine triphosphate (ATP), crystalline complexes and protein aggregates, mitochondrial dysfunction and lysosomal damage.NLRP3 activation by a signaling pathway that senses extracellular ATP or exposure to crystalline activators that mediate lysosome damage strongly induces the section of EV proteins,Mitochondrial dysfunction, characterized by dysregulated oxidative metabolism, is frequently associated with increased reactive oxygen species (ROS) production. ROS produced during oxidative phosphorylation represent a major endogenous source of genetic damage and mitochondrial mutations are linked to mitochondrial dysfunction, increased ROS production, and enhanced tumor proliferation and metastasis.A disturbance in ER homeostasis can promote in ER stress, which is characterized by disruption of normal protein folding events, decreased ER secretory function, and the accumulation of misfolded and unfolded proteins in the ER lumen, and results in the induction of the unfolded protein response. Notably, low nutrient and acidic pH conditions frequently found in tumor microenvironments can inhibit normal N‐linked glycosylation and side chain interactions to alter protein folding and activate the unfolded protein response.Mounting evidence suggests that inflammasome activity can regulate exosome secretion and composition, and that inflammasome activation can be induced by multiple stimuli associated with tumor development and exosome secretion. However, while these findings are suggestive, definitive studies have not been performed to address the relative contribution(s) of inflammasome activation in the regulation of TDE biogenesis and secretion.1.31.3.1Multiple studies now demonstrate that selective sorting processes regulate the accumulation of specific membrane and cytosolic proteins into developing ILVs during MVB biogenesis. Several reports indicate that the ubiquitination state of a protein, which is known to target both cytosolic and membrane proteins to early endosomes for subsequent degradation, is also an important determinant of selective protein accumulation into ILVs during MVB biogenesis. Further, it appears MVBs or a subset of ILVs within an MVB can be shunted to different pathways via a regulatory mechanism that are still not fully clear to target them to secretory or degradative fates.Mounting evidence also indicates that ESCRT complex proteins play an important role in sorting ubiquitinated proteins into developing ILVs. For example, the ESCRT‐0 protein HRS, which plays a key role in the initiation of ILV development, contains a ubiquitin interaction motif that promotes the accumulation of ubiquitinated proteins at clathrin‐coated membrane domains of early endosomes, and that mutations that block this ubiquitin‐binding activity also abolish the sorting function of HRS.1.3.2Exosomes carry cargoes that contain multiple distinct RNA species and are primarily enriched in small noncoding RNAs, including miRNAs, and such repertoires can markedly differ between exosomes isolated from normal and cancer cells of the same lineage.1.3.3Cancer‐associated mutant alleles represent obvious DNA biomarker targets for analyses of exosome cargoes, and in addition to their potential value as diagnostic and prognostic markers, may also provide critical information to inform treatment decisions. However, other exosome DNA parameters may also be informative. Exosomes derived from multiple different cancer types demonstrate a marked increase in overall DNA content,1.4Many of the factors involved in exosome biogenesis and secretion demonstrate a strong degree of evolutionary conservation.Enhanced exosome secretion rates observed from tumor cells appear to be induced in response to changes in cell phenotypes  and environmental conditions  that can also affect nonmalignant cells and tissues. It is less clear what specific processes regulate observed differences in the content of exosomes derived from malignant vs nonmalignant cells. Some of the enrichment observed in TDEs may reflect increased cellular abundance 2Recognition of tumor antigens by the immune system is essential for effective antitumor responses, and T‐cells reactive against specific tumor antigen‐derived epitopes are present both in the circulation and at tumor sites.Multiple immune cells are sensitive to TDEs‐mediated regulatory effects,2.1TDEs carry factors that can regulate innate and adaptive immune responses, and tumor‐associated antigens (TAAs) and immune‐regulatory molecules can be selectively packaged into TDEs by their parental tumor cells. Examples of exosome‐enrichment of TAAs include EGFR,2.2+ T cell‐dependent antitumor effects in a mouse tumor model.+ T‐cells and CD4+ T‐cells independent of MHC type, as demonstrated by cross‐protective reactivity against various mouse and human HCC cells.Several studies have shown that TDEs enriched in MHC‐I proteins or heat shock proteins (HSPs) represent a novel pathway through which tumor antigens can be transferred to DCs to prime T‐cell activation responses. When TDEs are loaded onto human DC cells, TDEs bearing MHC‐I proteins and the lysosome‐associated membrane protein‐1 were found to transfer tumor antigens, and to induce antigen‐specific CTL activation responses in vitro and induce CD8+ T‐cell responses, CD4+ T‐cell help, antigen‐specific antibodies, and a decrease in the tumor abundance of Tregs. These results imply that DC cells can capture and present tumor antigens more efficiently in vivo when they are presented in TDEs. However, the ability of TDEs to modulate antitumor immune responses may also depend upon TDE abundance, since glioma TDEs incubated with healthy donor peripheral blood mononuclear (PBMCs) can produce distinct immune effects depending upon the TDE concentration.Recent studies have compared the mode of tumor antigen capture by DC cells and whether the same antigen could induce variable antitumor effects when encountered through different delivery mechanisms. One study found that when cancer cells were engineered to express the same antigen on their cell surface, on their exosomes, or in a secreted form, the cells expressing the antigen on their exosomes induced a stronger antigen‐specific in vivo immune response.+ TDEs were also observed to stimulate NK cell migratory and cytolytic activity.HSPs are overexpressed in a variety of human cancers where they are associated with tumor development and metastasis, and are a common component TDE cargoes. TDEs derived from heat‐shocked mouse B‐lymphoma cells3TDEs have been reported to exhibit both negative and positive effects on immune responses, but the in vivo interactions and ultimate balance between these effects is not clear, and may depend upon the cells and tumor microenvironment from which a TDE population originates. Nevertheless, it seems clear that TDEs are capable of mediating multiple mechanisms that can promote tumor growth and metastasis by negatively regulating immune responses that would otherwise limit these phenotypes Figure .3.1+ T‐cell with its ligand PD‐L1 directly inhibits the effector functions of the CD8+ T‐cells by inducing regulatory pathways that control proliferation, survival, cytokine production, and cytotoxicity.The mechanisms that tumors employ to escape the negative regulatory effects of the host immune system have long been a subject of great interest.+ T‐cells , play key roles in immune homeostasis and self‐tolerance,+ T‐cell population when PBMCs are co‐incubated with TDEs, indicating that TDE exposure selectively impairs CTL effector cells (CD8+), without affecting the Treg population (CD4+). TDE exposure did not inhibit expression of the Treg marker FOXP3 and enhanced the ability of the exposed Tregs to respond to IL‐2 and inhibit T‐cell proliferation.Emerging evidence indicates that TDEs can induce Treg responses to promote Treg expansion and enhance their suppressor functions.Better understanding of the signaling pathways and factors that can converge to promote an immune response is an important challenge for developing effective immunotherapies. For example, both CTL and Treg responses are partially regulated by IL‐2 exposure, but Tregs respond to low IL‐2 concentrations that do not stimulate CTL responses, while CTL response tend to dominate at higher IL‐2 concentrations. Since TDE exposure can preferentially enhance Treg responses to IL‐2, administering IL‐2 to cancer patients with the goal of promoting CTL responses may promote a Treg response and the immune escape of the tumor rather than a tumor‐limiting CTL response.+CD25− T‐cells into CD4+CD25highFOXP3+ Treg.+ T‐cells and to activated CD4+ T‐cells.Circulating Treg concentrations are elevated in patients with head and neck cancer and ovarian cancer relative to healthy subjects, and serum from these patients are also highly enriched in TDEs.3.3+ T‐cells. These observations were explained in various cancers by a mechanism in which the presence of MHC‐I and a membrane‐associated form of FasL on the TDE surface could suppress T‐cell responses by inducing apoptosis,Early studies indicated that coculture of activated T‐cells with tumor cells, their supernatants or serum samples from cancer patients, but not with normal fibroblasts or their supernatants, could induce T‐cell apoptosis.+ T‐cells and it is clear that the Fas/FasL pathway is directly involved in spontaneous apoptosis of circulating Fas+ T‐cells.+ T‐cells in vivo through their expression of FASL , to increase their apoptosis rate.+ T‐cells by increasing CD3‐ζ and pSTAT5 expression. These results derive from studies performed with TDEs from head and neck cancer and melanoma. Additional studies should be performed to address the potential conservation of the mechanism among different tumor types, but it is evident that TDE‐induced tolerance plays an important role in the ability of tumors to escape the immune response.Expression of the T‐cell receptor (TCR) and its associated signal transducing CD3‐ζ chain is important for the functional integrity of the signaling pathway in T‐cells.Taken together, the listed TDE‐mediated effects that favor the development of immune tolerance responses, suggest that TDEs may be one of the driving forces that render tumor cells resistant to CTL attack.3.6As discussed above, TDEs can exert regulatory influences on immune responses through both local and systemic effects, and can carry factors that stimulate or suppress immune responses. However, perhaps the most important systemic role of TDEs appears to reside in their ability to build new niches that facilitate the development of tumor metastasis.44.1As previously discussed, the secretion of TDEs allow tumor cells to promote the apoptosis of activated T‐cells and NK cells to limit the ability of the immune system to attenuate tumor growth and progression by the depletion of tumor‐directed effector immune cells.4.2Monitoring Treg responses in cancer patients shows promise as a means to improve traditional cancer therapies and new cancer vaccines, as well to evaluate the potential for the development of recurrent disease. Treg frequency and suppressor function are significantly higher in patients receiving oncologic therapies than in those with active disease,4.3+ melanomas+ non–small‐cell lung cancer (NSCLC) tumors.Tumor‐specific immune responses can be induced by DC‐derived exosomes (DEX) after the DCs have been pulsed with tumor antigens+ TDEs induce more potent specific CTL responses than TDEs derived from cells without this IL‐18 modification.TDEs also demonstrate the ability to induce protective antitumor immunity and tumor‐specific T‐cell responses in vitro and in an in vivo animal model.For both the DEX and TDE vaccines the effort to improve their immunotherapeutic action should focus on enhancing the visibility of their presented to the immune system as a target for CTL and NK responses. While substantial research much still be conducted to determine if cancer vaccines based on DEXs or modified TDEs will be safe and effective, the initial results offer some encouragement. One critical concern for the potential of these approaches, however, arises from the fact that exosomes carry a broad array of factors that can influence cell phenotypes and great care must be taken to reduce the potential that such exosome‐based vaccine approaches do not also enhance the potential for subsequent or concurrent tumor metastasis by promoting the formation of premetastatic niches. This concern is exacerbated by our limited understanding of the systemic effects of specific exosome populations and the processes and underlying mechanisms that influence these effects. Given that most exosome vaccine studies have not achieved significant success and carry poorly understood risks, it may be premature to address the clinical potential of such approaches.5TDEs released from tumor cells not only alter the phenotype of adjacent normal cells and tumor‐infiltrating immune cells in the tumor microenvironment but also enter the systemic circulation to contact and alter the phenotype of distant cells, promoting the formation of distant premetastatic niches and altering the systemic immune repertoire to promote cancer development and metastasis. Mounting evidence indicates that changes in cancer cells and the tumor microenvironment can regulate the biogenesis, composition, and function of TDEs, however relative little is known about many of these processes, which could offer new avenues for tumor interventions. Indeed, at least one recent study has utilized a high‐throughput screening approach to identify the potential of repurposed drugs to act as selective inhibitors of exosome biogenesis.The tumor microenvironment regulates TDE production, immune cell infiltration, survival and activity, metastasis and chemotherapy resistance. Therapeutics that target tumor‐specific pathways that regulate these mechanisms offer another approach for cancer interventions. Most current research, however, has focused on employing TDEs in vaccine approaches designed to restore tumor‐specific immunity and thus promote tumor clearance. Such methods appear promising and have entered clinical trials, however, due to the potential of TDEs to inhibit antitumor responses and promote metastasis, substantial research needs to be performed to address safety concerns. Better insight into the interface between TDEs and innate and adaptive immunity could also provide novel tools to enhance antitumor responses. The heterogeneous origin and variable effects of TDEs represent a major challenge for future therapies aiming at silencing or modifying TDE effects on the immune system to promote tumor clearance. Substantial basic research and several clinical studies still need to be performed before currently proposed TDE‐based or TDE‐targeted therapies can be translated into clinical practice.Significant progress has been made in understanding the mechanisms that control exosome biogenesis, although most of this effort has focused on the study of factors and processes associated with ESCRT‐dependent exosome secretion. Much less is known about ESCRT‐independent exosome production, or how either process is regulated in response to environmental or malignant changes known to increase exosome secretion, or how these changes affect selective enrichment of TDE cargoes to mediate their activities. Better understanding of these topics are urgently required to permit the development of target approaches that can attenuate TDE secretion or TDE‐mediated systemic activities, while sparing potentially critical exosome activities regulated by nonmalignant tissues. Future therapeutics that attenuate TDE‐mediated effects appear likely to require targeted strategies to attenuate TDE secretion or selectively block the packaging of factors involved in key TDE‐mediated effects on tumor development or metastasis. The rapid growth in our knowledge of how exosome biogenesis and how TDEs differ from exosome produced by nonmalignant cells and tissues, offers hope that the discovery of candidate therapeutic TDE targets may be achievable within the near future.


GWS; exome or whole genome sequencing) is limited in British Columbia. The establishment of a translational research initiative (CAUSES) to provide diagnostic genome‐wide sequencing for 500 children necessitated the development of a genomic consultation service, a clinical service established to provide consultation for physicians considering GWS for their pediatric patients throughout British Columbia. The Genomic Consultation Service provides patient‐specific genomic advice that may include: GWS, multi‐gene panel, single gene test, referral to medical genetics for clinical evaluation, or no genetic testing. Here, we describe and evaluate this service.Access to clinical diagnostic genome‐wide sequencing  were processed in the first year. Only children were eligible for referral and average patient age was 8 years. Medical genetics was the most frequent referring discipline, followed by biochemical disease and pediatric neurology, respectively. Most patients (68%) had syndromic intellectual disability. There was a significant difference in the frequency of referrals not appropriate for GWS in the first versus the second 6 months of the service  suggesting increasing awareness of testing criteria by referring physicians.A total of 407 referrals  achieved in our  Although universal medical care is available through the provincial Medical Services Plan , funding for genetic testing is limited and is granted only in restricted situations. The CAUSES  Study was established as a 3‐year translational research initiative to perform diagnostic genome‐wide sequencing of 500 British Columbia children with suspected genetic disorders. In order to maximize patient benefits resulting from the study, a clinical Genomic Consultation Service was developed at BC Children's and Women's Hospitals, tertiary care centers providing academic clinical care to the province. The CAUSES Study (a trio‐based research study) considers patients for whom clinical diagnostic GWS is indicated and has a diagnostic rate of >40%  and the CAUSES research clinic were presented Figure .Following the initial meeting, each division appointed a representative who would serve as a spokesperson for their section, attend results discussions when the referring physician was not available, and bring suggestions and concerns to the Genomic Consultation and CAUSES Study teams. The Provincial Medical Genetics Program appointed a clinical geneticist who was independent of the CAUSES Study. A follow up education session was conducted for all appointed representatives where the CAUSES Study was explained in detail and the inclusion and exclusion criteria established for GWS were reviewed. Additional education sessions were also conducted including pediatric resident rounds and clinical genetics rounds  in order to inform potential referring clinicians.The Genomic Consultation Service meets weekly to triage and discuss approximately 10 patient referrals. Genetic counselors review each patient chart for detailed phenotypic information and lead the discussion of each referral with the team. The team is comprised of medical geneticists, a pediatric subspecialist, molecular geneticist, and genetic counselors. Advice on the most appropriate testing strategy is included in a letter to the referring physician for the health record. Referring physicians who disagree with the CAUSES team's decision can request reconsideration.2.2As the CAUSES Research Clinic provides trio‐based GWS, availability of both biological parents is required for enrollment. The inclusion criteria are as follows:Age ≤19 yearsPatient has a suspected single‐gene disorder plus one or more of the following:○Previous genetic investigations including chromosomal microarray analysis, appropriate single‐gene or available panel testing, and TIDE first tier biochemical testing for intellectual disability .Examples of circumstances when a patient would not be enrolled in the CAUSES Study include:an infectious, toxic or other nongenetic cause is likelya multifactorial condition is likelya teratogenic exposure is likelya well‐delineated chromosomal disorder was identified in the patienta Mendelian condition is suspected with limited genetic heterogeneity for which a targeted (and probably more cost‐effective) single‐gene test or gene panel is availablethe disease is likely to be caused by mutation of a novel human disease gene. .2.3We tracked the number of referrals, genomic advice provided, and the time required for review of patient records by the genetic counselors prior to the triage meeting and the time required for patient discussion.2.4This study was performed at the BC Children's and Women's Hospitals. The Research Ethics Board at UBC/BC Children's and Women's Hospital approved this study (H16‐02645).2.5Sex and the age of the patient were recorded. For analysis, age was divided into the following categories: Infant (<1 year); Preschool (>1–4 years); Elementary School (5–11 years); High School/Adolescent (12–19 years).2.6For purposes of analysis, the indications for diagnostic GWS were classified into four main categories:Isolated (nonsyndromic) Intellectual Disability—patients with intellectual disability and no major congenital anomalies.Syndromic Intellectual Disability—patients with intellectual disability and major malformations or multiple minor congenital anomalies or multi‐system medical issues.Multiple Congenital Anomalies without intellectual disability.Unexplained disorders of organ dysfunction without intellectual disability, patients with serious disorders of the blood, immune system, liver, kidney, endocrine system, neuromuscular function, or other organ system for which a genetic cause is suspected.2.7The clinical specialties and subspecialties of referring physicians were documented.2.8The genomic advice provided in a letter for the health record to the referring physician was classified under one of the following categories:Referral to the Provincial Medical Genetics Program for complete, in‐person clinical genetics evaluation is indicated;Targeted testing for a specific gene or available multi‐gene panel is indicated;GWS is not indicated for clinical purposes, but might be appropriate on a research basis through another study at our center or for investigation of a possible novel genetic cause;No further genetic testing is indicated at this time;Clinical GWS is indicated. If diagnostic GWS is clinically indicated, the referring physician is informed that she/he can request approval for this test through the Ministry of Health Out‐of‐Province Testing procedure or, if eligible according to the inclusion criteria described above, enroll the patient in the CAUSES Study;Defer—pending results of current testing;Decline for GWS through the CAUSES Study .2.9Chi‐square test (to compare sex of referred patients) and Fisher's exact test  were used as descriptive statistics  in addition to comparison of appropriateness for clinical GWS of Medical Genetics referrals versus all other disciplines for the first year.3During its first year, 407 genomic consultation referrals were processed . The lead genetic counselor required an average of 45 min to review each referral in detail and prepare it for presentation to the team. At the Genomic Consultation session, each case took approximately 7 min on average to discuss and determine the most appropriate genomic testing advice.p = .0006) were referred. The average patient age was 8 ± 5.1 years, with the largest age group 5–11 years (45%). The majority of referred patients had syndromic intellectual disability  and 238 males , followed by metabolic disease/biochemical genetics 21%) and neurology .The majority of referrals during the first year of operation of the service (71%) were appropriate for clinical GWS and the CAUSES Study. More referrals not appropriate for clinical GWS through the CAUSES Study were processed during the first 6 months 75/220, 34%) versus the second 6 months of the service .Within Canada, access to clinical diagnostic GWS is limited. The Genomic Consultation Service is designed to provide advice regarding appropriate genetic testing for pediatric patients being considered for diagnostic GWS. This service provides clinicians  an opportunity to interface with a genetics team and receive advice about genetic testing without having to wait many months for a full medical genetics consultation. This service is not a substitute for an in person consultation with a medical geneticist. The majority of patients were referred by medical geneticists in order to obtain diagnostic GWS that was not otherwise available to their patients. Many of the 40% of patients referred by other clinicians had previously been seen in medical genetics as well, which may explain why a medical genetics evaluation was suggested by the Genomic Consultation team in only 2% of referrals. There was not a significant difference between medical genetics and all other disciplines with respect to appropriateness of referral for diagnostic genome‐wide sequencing over the course of the year  and relevant previous investigations . This form was usually accompanied by the most recent and relevant consultation. Medical genetics consultations generally contained a comprehensive investigation list, but there was variability in the information provided from physicians. As a result, many referrals required extensive medical chart review. An electronic health record would have facilitated the time required for genetic counselor medical record review and preparation.Although genetic counselors have the training and expertise to triage genetic testing requests and identify the most appropriate genetic test for a variety of clinical indications, they are not always integrated into the process. Genetic counselors have also been used in the Center for Individualized Medicine at the Mayo Clinic to identify patients suitable for diagnostic GWS  to review and prepare referrals for patients being considered for genomic testing, particularly from nongeneticist specialists. Utilization of genetic counselors in the triage processes for genetic testing has been shown to have substantial cost efficiencies. A case study involving one genetic services laboratory demonstrated that integration of genetic counselors into the triage process generated cost savings of more than $1,000,000 through the cancelation of misordered tests over 21 months , an application is submitted directly to the Ministry of Health  with justification . In other provinces, there are institutional committees that determine whether GWS will be funded. The concept of an institutional gatekeeper has been discussed  determined by our group were consistent with the literature and were described in education sessions and correspondences to referring physicians. Education of referring physicians with ongoing communication is an important consideration with clinical implementation of GWS . This difference may be due to learning and increased awareness of patient suitability by referring physicians.The Genomic Consultation team's decision followed the inclusion/exclusion guidelines with the primary consideration being the likelihood of identifying an underlying Mendelian disorder(s). The need for appropriate initial investigations  was transmitted to physicians as part of the educational sessions, and only 1% of total of referrals were deferred due to inadequate clinical information/investigations. Moreover, while approximately 11% of patients were declined for clinical GWS in the CAUSES Study due to lack of suitability of referral, more referrals were deemed inappropriate for clinical GWS during the first 6 months (75) than in the second 6 months of the service (42)  is not surprising. Idiopathic developmental delay/ID is the most common pediatric referral to medical genetics and is an indication for GWS (Deciphering Developmental Disorders Study, The Genomic Consultation Service likely contributed to improving the diagnostic yield and appropriate use of GWS in our patients suspected of having rare genetic diseases. The Genomic Consultation Service was utilized by physicians throughout British Columbia who were considering GWS for their patients. We did not demonstrate a significant difference between referrals from medical genetics versus other disciplines for appropriateness for clinical GWS and the CAUSES Study. We have also demonstrated that appropriate patient selection through this service improves the cost efficiency of delivering a diagnostic GWS program (Dragojlovic et al., None declared.


Ecologists are pressed to understand how climate constrains the timings of annual biological events (phenology). Climate influences on phenology are likely significant in estuarine watersheds because many watersheds provide seasonal fish nurseries where juvenile presence is synched with favorable conditions. While ecologists have long recognized that estuaries are generally important to juvenile fish, we incompletely understand the specific ecosystem dynamics that contribute to their nursery habitat value, limiting our ability to identify and protect vital habitat components. Here we examined the annual timing of juvenile coldwater fish migrating through a seasonally warm, hydrologically managed watershed. Our goal was to (1) understand how climate constrained the seasonal timing of water conditions necessary for juvenile fish to use nursery habitats and (2) inform management decisions about (a) mitigating climate‐mediated stress on nursery habitat function and (b) conserving heat‐constrained species in warming environments. Cool, wet winters deposited snow and cold water into mountains and reservoirs, which kept the lower watershed adequately cool for juveniles through the spring despite the region approaching its hot, dry summers. For every 1°C waters in April were colder, the juvenile fish population (1) inhabited the watershed 4–7 d longer and (2) entered marine waters, where survival is size selective, at maximum sizes 2.1 mm larger. Climate therefore appeared to constrain the nursery functions of this system by determining seasonal windows of tolerable rearing conditions, and cold water appeared to be a vital ecosystem component that promoted juvenile rearing. Fish in this system inhabit the southernmost extent of their range and already rear during the coolest part of the year, suggesting that a warming climate will truncate rather than shift their annual presence. Our findings are concerning for coldwater diadromous species in general because warming climates may constrain watershed use and diminish viability of life histories  and associated portfolio benefits over the long term. Lower watershed nurseries for coldwater fish in warming climates may be enhanced through allocating coldwater reservoir releases to prolong juvenile rearing periods downstream or restorations that facilitate colder conditions. Many taxa migrate to track favorable conditions that vary in time and space. Reproduction is often timed in migratory life histories so that juveniles can exploit conditions that promote growth and survival , an anadromous species that rears in steams, floodplains, and estuaries of the Pacific Rim .available online).Data describing monthly mean air temperatures and precipitation were provided by a NOAA weather station near the Sacramento River .,Data describing daily water flow were provided by U.S. Geological Survey gages on the Sacramento and San Joaquin Rivers to quantify the magnitude of annual high flow events (data available online).Data describing fish habitat temperature and fish presence were provided by the U.S. Fish and Wildlife Service. The Service concurrently monitors water temperatures and juvenile salmon throughout the Sacramento River and Delta via point measurements , and we applied a log‐transformation to normalize the distribution of these data.We used temperature data collected during beach seining and trawling to quantify for each water year indexes of temperatures during April in the Sacramento River and Sacramento–San Joaquin Delta  were often heavily influenced by smaller numbers of migrants observed late in the summer. We combined all measurements taken alongshore of each region  whereas these conditions in mid‐channel waters were described at one location; thus, habitat conditions and fish responses along shorelines were summarized from spatially aggregated data describing a region and in mid‐channel waters they described a single station where fish were presumably leaving these regions.and water flow, but these variables confounded models because they were correlated (r2 = 0.73 [Sacramento River (Sac. R.) shore], 0.70 [Delta shore], 0.70 [Sac. R. mid‐channel], 0.38 [Delta mid‐channel]). We therefore modeled juvenile salmon responses to temperature alone, as temperature is particularly well known to impact salmon in this system . There was a frontier of maximum lengths in salmon emigrating to sea given the date, and this frontier contracted to exclude larger fish in years with warmer springtime waters  to enter marine waters in the winter would presumably be less impacted by warm springs. Secondly, we chose broad‐scale metrics to describe our study system. Fish experienced a more nuanced, dynamic environment beyond what we could measure that depended on finer‐scale habitat conditions and fish movements. An example of this supported by our data is that fish may use shoreline waters until they exceed tolerable levels and then retreat to cooler mid‐channel waters before leaving the system entirely. In addition, metrics that described environmental conditions in certain months were probably correlated with those of proximate months and our models are probably measuring their response to both. However, that our model understanding of the environment correlated well with fish responses suggests that we have parsimoniously captured the phenomenon: cold, wet winters keep waters cool and flowing high longer, allowing fish to depart to sea later and larger. Finally, we may expect that, compared to more natural systems, our system's lack of juvenile age structure  and habitat complexity  may contribute to an especially apparent, population‐level phenological response of fish to temperature.Our study would also be enhanced by a greater understanding of habitat use in mid‐channel waters and outcomes  of populations that departed earlier and smaller. In contrast to measurements of habitat use along shore, fish observations in mid‐channel waters only occurred in two locations. This likely limited our understanding of the temperatures that fish select for because temperatures are likely to vary substantially among locations in the watershed and may explain why, in contrast to habitat use in shoreline waters, we did not detect fish in mid‐channel waters using cooler than average temperatures in the spring. Understanding the demographic consequences  of reductions in outmigration windows and maximum outmigration sizes would further improve the application of this work for identifying the relative benefits of water management. For example, departure timing may reflect mortality as well as higher and earlier emigration rates in warm years and it would be informative to quantify relationships between fish size at emigration and survival or reproductive success at later life stages  may portend poor nursery habitat conditions for fish that year. This is significant because many species rely on freshwater and estuarine waters during critical juvenile phases (Beck et al. However, ecologists and managers are developing more sophisticated and nuanced approaches to water regulation in conservation contexts (Danner et al.  Click here for additional data file. Click here for additional data file.


Aging inevitably leads to reduced immune function, leaving the elderly more susceptible to infections, less able to respond to pathogen challenges, and less responsive to preventative vaccinations. No cell type is exempt from the ravages of age, and extensive studies have found age‐related alterations in the frequencies and functions of both stem and progenitor cells, as well as effector cells of both the innate and adaptive immune systems. The intrinsic functional reduction in immune competence is also associated with low‐grade chronic inflammation, termed “inflamm‐aging,” which further perpetuates immune dysfunction. While many of these age‐related cellular changes are well characterized, understanding the molecular changes that underpin the functional decline has proven more difficult. Changes in chromatin are increasingly appreciated as a causative mechanism of cellular and organismal aging across species. These changes include increased genomic instability through loss of heterochromatin and increased DNA damage, telomere attrition, and epigenetic alterations. In this review, we discuss the connections between chromatin, immunocompetence, and the loss of function associated with mammalian immune aging. Through understanding the molecular events which underpin the phenotypic changes observed in the aged immune system, it is hoped that the aged immune system can be restored to provide youthful immunity once more. These c2.1Hematopoietic stem cells (HSCs) are responsible for the maintenance and replenishment of immune and hematopoietic cells throughout life. HSC numbers actually increase with age, both in mice and humans, but these accumulated cells show reduced reconstitution potential  diversity shrinks with age in both CD4+ and CD8+ compartments, and inequalities in clonal size may compromise immunity or even lead to autoimmunity  family members , evidence suggests that individuals with a higher proportion of innate‐like T cells are less likely to exhibit frailty and immune deterioration  and a commensurate reduction in classical monocyte (CD14+CD16‐) frequency in elderly individuals  cells is maintained or even increased in old age but NK functional capacity, such as cytotoxicity and cytokine secretion, decreases leaving older individuals vulnerable to tumors and infections , chromatin structure and epigenetic modifications have critical roles in all aspects of DNA‐related processes including transcription, replication, and repair  and chromatin state  as mice or human age , are canonical marks of repressed chromatin, whereas others, such as trimethylation of lysine 4 of histone‐H3 (H3K4me3), are associated with open chromatin and active transcription.Post‐translational modifications to the + T‐cell effector responses to Listeria monocytogenes infection together with a defect in the silencing of stem‐related and memory‐related genes (Pace et al., Very few studies have been conducted to date examining age‐related genome‐wide changes in histone modifications in mammalian cells, let alone cells of the immune system. One study examining epigenetic changes in old and young mouse HSCs found increased H3K4me3 peaks in the promoters of genes associated with HSC identity and self‐renewal, and conversely increased DNA methylation at transcription factor binding sites in differentiation‐promoting genes (Sun et al., + T cells is altered with age (Moskowitz et al., + T cells from older humans showed a loss of chromatin accessibility at gene promoters targeted by the transcription factor NRF1 (Moskowitz et al., + T‐cell compartment in old age. Another study found that chromatin in human PBMCs from older individuals was more closed at promoters and enhancers associated with T‐cell signaling, such as the IL‐7Rα gene as previously mentioned, compared to that seen in younger individuals (Ucar et al., Recent studies examining chromatin accessibility using ATAC‐seq support the idea that the epigenetic landscape of CD8C. elegans and may well have a role in immune aging.There are a plethora of other chromatin‐modifying proteins which impart and remove a wide variety of histone modifications but which have not been examined in the context of immune aging. A study recently published used a single cell mass cytometry approach to provide the first insight of the effect of aging on 40 of these marks in different human immune cell populations, revealing more variation in chromatin modification profiles in older individuals compared to younger individuals (Cheung et al., Increased levels of H4K20me3 have been found in the liver and kidneys of aged rats (Sarg, Koutzamani, Helliger, Rundquist, & Lindner, Bach2 locus has been linked to CD4+ T‐cell senescence (Kuwahara et al., INK4A‐ARF locus, which leads to cellular senescence and aging of a variety of cell types including mouse HSCs (Janzen et al., Dysregulation of histone post‐translational modifications does not need to occur genomewide to have marked influences on cellular function as changes in the epigenetic regulation of key genes can lead to cellular senescence and aging. For example, decreased histone acetylation at the mouse INK4A‐ARF locus, the p53 tumor suppressor, and the histone demethylase Jumonji D3 may also occur as this has been reported in mouse neural stem cells (Sola et al., INK4A‐ARF locus leads to expression of the ARF protein which causes nuclear accumulation of p53 and increased transcription of the p21 gene, a cyclin‐dependent kinase inhibitor which induces cellular senescence (Ene et al., INK4A, another protein encoded in the INK4A‐ARF locus, has been proposed to have utility as a biomarker of aging when measured in human peripheral T lymphocytes (Y. Liu et al., Complex interplay between the ARF protein encoded by the 4As summarized above, there is no doubt that aging is both associated with alterations in immune cell frequency and function, as well as a suite of chromatin changes in immune and non‐immune cell types. Determining causality between these two phenomena is somewhat more difficult. Chromatin alterations observed with age may drive the cellular dysfunction, or alternatively, cellular dysfunction may cause chromatin alterations, or perhaps both (see Figure Regardless of whether chromatin alterations or cellular phenotype or environment are considered the “cause” of the differential effects of aging, if the phenotype can be rescued through modulating chromatin‐modifying enzymes then the chromatin alteration can surely be viewed as the molecular driver of age‐related immune dysfunction. This is clearly the case when telomerase activity is restored (Bodnar et al., 5The quest for a central unified theory of aging has revealed several molecular hallmarks common across cell type and species. We can infer a lot from studies in model organisms; however, direct evidence demonstrating the molecular basis of cell type‐specific dysfunction within the human immune system remains scarce. Part of the reason for this may be the technical limitations of performing genomewide epigenomic analysis on limited starting material, such as that required for human samples and smaller cellular populations from mice and other non‐human mammals. Rapid technological development including the advent and more wide‐spread use of single cell RNA‐seq and ATAC‐seq methodologies, and advances in ChIP‐seq‐based assays, mean molecular analyses of small cell numbers is now more achievable than ever. Applying these technological advances to the phenotypic changes observed in the aged immune system will rapidly advance this field and hopefully reveal key molecular events which underpin the loss of immune function in old age. Through this approach and the burgeoning field of epigenetic drug discovery (Tough, Tak, Tarakhovsky, & Prinjha, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Both authors have made substantial, direct, and intellectual contribution to the work and approved it for publication.


Lancet editorial observed that “the last big shared challenge remaining is testing—in every region the number of undiagnosed HIV infections remains a substantial barrier to achieving UNAIDS targets and ending AIDS by 2030” Globally, we are at an inflection point in achieving UNAIDS’ 95‐95‐95 goals for 2030. A recent ® HIV Self‐Test Kit introducing the first HIV rapid test kit intended for use by the general population and available for purchase over‐the‐counter in the United States. Building on a history of public health interventions aimed at self‐screening for health conditions that includes home pregnancy tests, breast self‐examinations for cancer screening and blood‐glucose monitoring, access to HIVST permits individuals perceiving themselves to be at‐risk of infection to test independently and privately. Global attention to the potential of HIVST took root in 2013 following the OraQuick FDA approval, with UNAIDS and the World Health Organization (WHO) holding an initial consultation on the ethical and public health implications of HIVST. At that time, no HIVST kits were publicly available in low‐ and middle‐income countries (LMIC) , no normative guidelines had been established and only a small body of LMIC‐focused evidence around HIVST existed. Within a year, a full HIVST journal supplement had been published exploring early issues in HIVST introduction: regulation and policy, optimal product profiles, ethical considerations and both positive and negative potential impacts of rollout HIV self‐testing (HIVST) has developed substantially in recent years and is now considered a new and critical HIV response strategy in controlling the epidemic. In 2012, the US FDA approved the OraQuickThese initial supportive efforts established the foundation needed for HIVST expansion across countries. The second three‐year phase of STAR added Lesotho, Eswatini and South Africa, and aimed to create a market for HIVST and evaluate optimal distribution models for increasing access to testing among those unwilling or unable to utilize traditional testing venues and ensuring linkage from a preliminary positive HIVST result to confirmatory testing and treatment. By the end of the programme, Unitaid, the STAR programme's commodities funder, will have provided five million HIVST kits to the six project countries. Building on STAR's momentum and their own smaller scale pilot programmes in 2016, PEPFAR expanded its HIVST programming and will have delivered 2.3 million HIVST kits across 11 countries in 2017 to 2018. Similarly, the Global Fund is expanding HIVST support across 18 countries, estimated to cover about 12% of the global HIVST volume Supplement present a diverse range of the key findings from the first phase of STAR, and provide a basis for needed programmatic action to accelerate expansion. Presently, in sub‐Saharan Africa, there are 15 countries that have HIVST policies in place or under consideration and multiple products available with some type of certification et al. explore the regulatory environment in Malawi, Zambia and Zimbabwe in vitro diagnostic tests in general, and HIVST kits were no exception. The authors call for a regional collaboration to spread the regulatory burden across countries and facilitate the passing of required legislation to support more codified regulation of diagnostics.The articles collected for this Supplement, Simwinga et al. present findings from Malawi and Zambia evaluating an IFU translated into the local language and evaluated for clarity and ease of use WHO prequalified test kits have gone through quality assurance evaluations aimed at ensuring “global standards of quality, safety and efficacy” to support Ministries of Health and the introduction of quality diagnostics et al. evaluated the OraQuick HIV‐1/2 antibody test kits for result stability post‐testing. They showed that while strongly reactive HIVST remained stable, 29% of initially non‐reactive kits converting to be weakly reactive false positive when read at least four days later, countering previous work which indicated OraQuick test kits were stable for up to a year against any delayed readings of kits In another study, given that programmes have proposed late reading of returned kits to determine HIV positivity, Watson et al. model how HIVST and other “test for triage” strategies might impact national algorithm performance Eaton Supplement, Sibanda et al. began with the clients, investigating preferences for access to HIVST in rural Zimbabwe through discrete choice assessments, finding respondent preferences for door‐to‐door distribution, kits free‐of‐charge, access by telephone to help in using kits and linkage to confirmatory testing, and that programmes use patient reminders and outreach to enhance effectiveness Methodologies for HIVST distribution will be a critical aspect of programme effectiveness. Various delivery modes have been considered: vending machines, over‐the‐counter at pharmacies, secondary distribution when an HIVST is distributed to one person for use by another, and facility‐ and community‐based distribution Supplement, Hatzold et al. reviewed STAR data from Malawi, Zambia and Zimbabwe that assessed the integration of HIVST tools into HIV programming Also in this Supplement, Kumwenda et al. provide new evidence on social harms from projects in Malawi, summarizing data from six HIVST projects from 2011 to 2017 where a combination of qualitative and quantitative methods were used Advantages of HIVST, such as the ability to test privately, may also be misused or abused and potential social harms should not be ignored. Previously in Kenya, low rates of physical and verbal abuse have been reported with the introduction of HIVST kits by women for their male partners to test themselves Supplement, Neuman et al. reflect on the difficulties in estimating linkage as HIVST is brought to scale In the context of HIVST, linkage to care refers not just to the initiation of ART, but first to confirmatory testing after a positive HIVST Supplement, Mangenah et al. performed a cost analysis of community HIVST kit distribution in Malawi, Zambia and Zimbabwe as well as a sensitivity and scenario analysis to project future costs et al. use this data to present a modelling analysis comparing community distribution to three priority populations in Zimbabwe and Malawi: women having transactional sex (WTS), youth and adult men et al. have added to cost‐effectiveness research, considering the trade‐offs between investing in HIVST and other HIV programmes, they estimate that HIVST is cost‐effective when the mean cost per disability adjusted life year averted is below US$ 500. According to their models, this occurs when HIVST kits were distributed to WTS and men but not to youth.Costing HTS is highly contextual with considerable variation, but important to programme planning and bringing HIVST to scale. In this et al. focus on the broader policy environment in LMIC, market development for HIVST kits, the STAR programme experience and its positioning for HIVST scale‐up et al. also touch on outstanding challenges to be addressed and present a call to action to maintain momentum in bringing HIVST to scale Offering commentary on the use of HIVST, Ingold Since 2012, substantial progress has been made on HIVST programmes, policy and products – but few countries are implementing HIVST at scale, with many still conducting smaller volume pilot programmes. Recent HIV response framings have declared that “what got us here won't get us there” However, operational questions remain. Intentional misuse, accuracy and performance of secondary distribution, more effective leveraging of public–private sector collaborations to reach high‐risk populations, programmatic use of blood‐based HIVST kits, use of HIVST as a demand generation tool for Pre‐exposure prophylaxis (PrEP), volume procurement approaches to reduce unit pricing and the use of mobile technology and other methods to estimate linkage post‐HIVST, could all benefit from more operations research to guide programming.Supplement adds significantly to the field of HTS, exemplifying the potential public health role of this new technology to critically increase coverage. Still, HIVST access will not reach the scale needed to impact the epidemic without both leveraging existing health programmes and developing new and innovative avenues of access. The integration of HIVST should work to amplify existing HIV programming to achieve multiple purposes that serve public health goals: reaching unreached and high‐risk individuals at an early disease stage, reducing testing burdens on taxed health systems, and critically identifying the most effective avenues to linking persons screening positive to onward testing and treating or linking persons screening negative to prevention services. All are outcomes of critical importance to controlling the HIV epidemic. And novel avenues such as private sector delivery will continue to need to be explored. While we find ourselves at the “last big shared challenge” of HIV testing, in the race towards control of the HIV epidemic, this Supplement strongly illustrates that a new testing paradigm based in part on HIVST is key to the next decade of the HIV response and achieving 95% diagnosis rates everywhere.The body of evidence produced in this VW, EJ, NF and HI declare no competing interests.VW and EJ drafted the initial manuscript. All authors critically reviewed the manuscript, suggested revisions and editorial changes, and approved the final version.VW is a member of the Unitaid HIVSTAR Technical Advisory Group.