The aim of this study was to examine continuation of subcutaneous and intramuscular depot medroxyprogesterone acetate (DMPA-SC and DMPA-IM) when administered by facility-based health workers in Burkina Faso and Village Health Teams (VHTs) in Uganda.Participants were family planning clients of health centers (Burkina Faso) or VHTs (Uganda) who had decided to initiate injectable use. Women selected DMPA-SC or DMPA-IM and study staff followed them for up to four injections (providing 12 months of pregnancy protection) to determine contraceptive continuation. Study staff interviewed women at their first injection (baseline), second injection, fourth injection and if they discontinued either product.N=990, 492 DMPA-SC and 498 DMPA-IM). Twelve-month continuation in Uganda was 77.8% for DMPA-SC and 77.4% for DMPA-IM . Reasons for discontinuation of DMPA across groups in Burkina Faso included side effects , being late for injection  and refusal of spouse . Reasons for discontinuation in Uganda included being late for injection , received from non-VHT  and side effects . Increased age  and partner acceptance of family planning  had protective effects against discontinuation in Burkina Faso; we did not find statistically significant variables in Uganda.Twelve-month continuation in Burkina Faso was 50% for DMPA-SC and 47.4% for DMPA-IM  between DMPA-SC and DMPA-IM whether from facility-based health workers in Burkina Faso or VHTs in Uganda. Continuation was higher through community-based distribution in Uganda than health facilities in Burkina Faso.The subcutaneous formulation of depot medroxyprogesterone acetate (DMPA-SC) is increasingly available in Family Planning 2020 countries. Use of DMPA-SC does not appear to change continuation relative to traditional intramuscular DMPA. Growing evidence of DMPA-SC's suitability for community-based distribution and self-injection may yield indirect benefits for contraceptive continuation and help reach new users. The contraceptive prevalence rate in Burkina Faso is low (around 15% of women of reproductive age are using contraceptive methods) and has been slow to increase Contraceptive use is higher in Uganda , with injectables being the most commonly used method among married and unmarried women Addressing unmet need for family planning requires supporting women to continue using contraception for as long as they wish to do so, in addition to reaching new users of contraception 22.1In Burkina Faso, we conducted the study in four regions . Study participants were newly initiating injectable contraceptive users from 10 participating health facilities in each of the 4 regions. Of 40 participating facilities, 31 were Centres de Santé et de Promotion Sociale, the most peripheral level of medical care available, serving the most remote areas of Burkina Faso and covering a population of 8000 to 15,000. Seven facilities in the study were nongovernmental organization (NGO) family planning clinics administered by either Marie Stopes Burkina Faso or L'Association Burkinabè pour le Bien-Être Familial and located in the larger cities of Ouagadougou, Bobo Dioulasso and Koudougou. The remaining two study sites were larger medical centers.In Uganda, we implemented the study through VHTs  in six districts . A total of 27 health facilities participated , with the affiliated VHTs recruiting community-based clients who were newly initiating injectable contraception into either the DMPA-SC or DMPA-IM group.In both study settings, we carried out a prospective cohort study. Women seeking injectable contraception at a participating site self-selected DMPA-SC or DMPA-IM. All health workers involved in the study counseled women on both injectables and provided DMPA-SC or DMPA-IM based on the client's preference; study staff provided additional training and supervision to minimize the potential for provider bias. After giving the injectable contraceptive to the woman, the health worker asked her interest in participating in the study using a recruitment script and eligibility checklist. A woman was eligible if she was 18 to 49 years old (15 to 49 in Uganda), had no contraindications to DMPA, was newly initiating the injectable at the time of enrollment (either a new user of injectables or a woman who had used injectables in the past but had stopped), planned to reside in the area for the 12 months, expressed a desire to prevent pregnancy for at least 12 months and provided informed consent. Study staff noted the injection schedule for each woman so that research assistants could plan to return to interview the women at specific time points. Research assistants then followed up with eligible women who had indicated a willingness to participate, explained the study further, and enrolled them as a DMPA-SC or DMPA-IM user in the study if they agreed to participate. Enrollment in both studies began in December 2015 and continued through April 2016.Research assistants followed participants for up to four injections (providing approximately 12 months of pregnancy protection), concluding in April 2017. We describe more information on follow-up procedures in 2.2The Ministry of Health Comité d'Ethique pour la Recherche en Santé approved the Burkina Faso study on October 7, 2015. The Higher Degrees, Research and Ethics Committee of Makerere University College of Health Sciences School of Public Health and the Uganda National Council for Science and Technology approved the Uganda study on November 2, 2015.2.3We calculated sample sizes with the intent to observe whether or not there was a significant difference in 12-month injectable continuation use between DMPA-IM and DMPA-SC users. The target sample size for the Burkina Faso study (483 women per group) assumes a statistical power of 90%, a significance level of .05 and a 10-percentage-point difference in continuation rates between DMPA-IM users (an estimated 72% The target sample size for the Uganda study (604 women per group) assumes a statistical power of 87%, a significance level of .05 and a 10-percentage-point difference in continuation rates between DMPA-IM users  and performed detailed follow-up surveys at the second and fourth injections or upon discontinuation. Health providers kept track of whether women enrolled in the study returned for their second, third and fourth injections on time, and alerted research assistants about which participants returned for their reinjections within the 30-day window and which did not. Research assistants then proceeded to contact participants, either by phone or in person as preferred by the participants at baseline, to conduct the follow-up or discontinuation surveys (this only occurred after the 30-day window for reinjection had closed).Follow-up interviews documented women's second and fourth injections and asked women about their experience and satisfaction with either method. At 6 months, if women received their third injection, research assistants noted this, although they did not conduct interviews in order to preserve women's time. Women who stopped the injectable or who received injections after the close of a 30-day injection window were asked to respond to a discontinuation survey. Discontinuation surveys asked participants about their reasons for discontinuation, including their personal satisfaction with the chosen method, any changes to their lifestyle that occurred and practical issues that are common barriers to continuation . Special 3- and 9-month surveys were created for women who switched from one DMPA injectable to the other.2.5t test for continuous covariates and χ2 tests for categorical covariates. Second, we calculated continuation rates of DMPA-SC and DMPA-IM as the proportion of women who completed four consecutive injections providing 12 months of pregnancy protection. Women who did not receive their second, third or fourth injection were considered discontinuers. We calculated continuation rates based on the principle of intention to treat. For the analysis, we classified women lost to follow-up as discontinuers in their original group and women who switched injectable types during the study as continuers in their original injectable group. We also assessed the probability of continuation over four injections using a Kaplan–Meir estimator, also known as a survival analysis. We used the time to discontinuation as the outcome variable for the survival analysis. Finally, we ran Cox proportional-hazards regression models to determine the factors associated with the risk of discontinuation of DMPA. We chose factors to include in the final Cox hazard models based on significance in crude hazard ratios for discontinuation and theoretical associations with discontinuation of injectable contraceptives in these settings.First, we assessed baseline characteristics of study participants for comparability between DMPA-SC and DMPA-IM groups using 33.1In Burkina Faso, 492 women enrolled in the DMPA-SC group and 498 in the DMPA-IM group. There were several statistically significant differences between groups; women who chose DMPA-SC were slightly younger, had lower parity and were more likely to currently be in school. In addition, women who chose DMPA-SC were less likely to be new users of family planning and were more likely to have previously used injectable contraception. Lastly, DMPA-SC users reported a higher number of household assets .Table 1DBaseline characteristics were similar between DMPA-IM and DMPA-SC groups in Uganda, although DMPA-SC users were more likely to report that they made family planning decisions jointly . Across We describe the number of women contacted, enrolled in each group and included in the analysis in 3.2n=990): 11 in the DMPA-SC group and 8 in the DMPA-IM group. In all, 50 women switched from DMPA-SC to DMPA-IM, and 19 women switched from DMPA-IM to DMPA-SC (two women switched at both the second and fourth injections). Overall, 50.0% of the DMPA-SC group continued for four injections (approximately 12 months) compared to 47.4% of the DMPA-IM group : 21 in the DMPA-SC group and 24 in the DMPA-IM group. A total of 36 women switched from DMPA-SC to DMPA-IM, and 79 women switched from DMPA-IM to DMPA-SC (9 women switched at both the second and fourth injections). Overall, 77.8% of the DMPA-SC group continued through four injections compared to 77.4% of the DMPA-IM group  to the group to which they switched to, the continuation rates remained similar, and the difference was nonsignificant.3.3In Burkina Faso, when considering multiple factors that could be linked to discontinuation, under the hazard ratio model, age was a moderate risk factor for discontinuation; as age increased, the risk of discontinuation decreased by 2% . Partner acceptance of family planning was highly predictive of discontinuation. Having a partner who approved of contraceptive use decreased the risk of discontinuation by 52% even when controlling for other factors in the multivariate hazard ratio model  .Table 2CIn Uganda, no single factor predicting discontinuation remained significant when adjusted for other variables despite many crude hazard ratios that were statistically significant .3.4The most commonly reported principle reasons for discontinuation of DMPA in Burkina Faso were side effects (18.3%), being late for injection (13.8%) and refusal of spouse (10.4%). Reasons for discontinuation were similar across groups . A total4These findings contribute to emerging literature on contraceptive discontinuation, specifically adding the first-ever data on women's continuation of the more recently introduced injectable contraceptive, DMPA-SC, compared to the more traditional injectable, DMPA-IM, when both products are administered by health workers. Given a new 6-year agreement announced in 2017 that guarantees near parity in price between DMPA-IM and DMPA-SC for qualified buyers , there is even greater interest in understanding the potential benefits of this new product option. Overall, these injectable continuation studies in Burkina Faso and Uganda indicate that the two DMPA injectable types are likely to have very similar continuation rates when delivered by the same health workers. At the same time, due to the product's ease of use, introduction of DMPA-SC holds particular potential to catalyze investments in and use of delivery options such as community-based distribution and self-injection of contraception, and increase access for women.This study also yields insights relevant to delivery of injectable contraception more generally and links to contraceptive continuation. According to a multicountry analysis, the median duration of injectable contraceptive use is 11.9 months — lower than for IUDs, condoms, pills and traditional methods — and the probability of continuation of injectable contraception at 12 months is 59.4% Information from these studies on women's reported reasons for discontinuation suggests that we can do more to support women who wish to continue using contraception for longer periods of time. Similar to previous findings that many women discontinue injectables for method-related reasons (such as side effects or myths) Studies have demonstrated that the efficacy, safety and immediacy of contraceptive effect of DMPA-SC are equivalent to DMPA-IM; tolerability of the product is generally expected to be comparable as well. Perhaps, then, the lack of continuation differences when the same health workers deliver the two products is not surprising. At the same time, previous research shows that injectable contraception users and health workers, including CHWs, in Uganda and Senegal preferred DMPA-SC over DMPA-IM 4.1We excluded women already using injectable contraception from participation in order to calculate continuation from the first injection for all women; therefore, our samples may not be representative of injectable contraceptive users in these countries. Additionally, we designed these studies to compare continuation between two DMPA injectable products; some discontinuers switched to other contraceptive methods, so DMPA discontinuation may not translate to changes in broader measures like contraceptive prevalence rates and unmet need for family planning.


Evidence of involvement of novel biomarkers in disease pathogenesis from research cohorts often precedes an understanding of their distributions in broader populations. This study aimed to estimate the distribution of fibroblast growth factor 23 (FGF-23), an endocrine hormone that helps to regulate serum phosphate levels, in the overall US population and in important subgroups. We used a predictive model generated using data from the Framingham Health Study to estimate FGF-23 values for adults in the US National Health and Nutrition Examination Survey and the size of patient subgroups with levels of FGF-23 above selected thresholds. To assess the face validity of our FGF-23 estimates, we examined the relationship between estimated FGF-23 and cardiovascular and all-cause mortality within NHANES using Kaplan-Meier estimates and Cox proportional-hazards regression models and compared it to that observed in Framingham. Estimated FGF-23 values from NHANES were lower  than the observed FGF-23 values from the Framingham cohort. Age- and sex-adjusted 10-year all-cause mortality was significantly higher  for subjects with estimated FGF-23 levels in the highest versus lowest quartile. Estimating the distribution of biomarker values in the general population by applying predictive equations from smaller research cohorts is feasible and can inform drug research decision making. The development of new medicines is often aided by the exploration of novel laboratory biomarkers and the role that they play in disease pathogenesis. Such biomarkers can inform the drug development process in several ways. Early on, they provide an invaluable tool for scientists working to elucidate disease pathways, identify treatment targets, and test the effects of potential drug candidates in pre-clinical disease models. Later, as pharmacodynamic measures, they can enable demonstration of proof of concept in early phase clinical research studies, pending confirmation of efficacy on more clinically meaningful patient outcomes in large Phase 3 clinical trials . FinallyEvidence of the association between a novel biomarker and the unfolding of disease in select cohorts often precedes the understanding of the prevalence of the biomarker in broader populations in which it may ultimately be used. For example, while findings from the Framingham Health Study (FHS)  provided compelling evidence that elevated cholesterol was associated with coronary heart disease as early as 1961 , an asseGiven this time lag in understanding biomarker epidemiology, drug development decisions are often made based on incomplete information about the extent to which abnormalities seen in small cohort studies translate to the overall disease population. With the growing availability of large, “real-world” datasets and the increasing potential for linkage between datasets with disparate data elements, the potential exists to leverage linked data to produce estimates of biomarker prevalence and hence the size of a potential disease population to target with a novel treatment directed at a specific mechanism reflected by that biomarker. In addition, such estimates can be used to partially validate biomarker-disease associations seen in convenience samples using larger datasets.In the present study, we demonstrate this approach using the example of fibroblast growth factor 23 (FGF-23), an endocrine hormone primarily produced in bone that helps regulate serum phosphate levels by inducing renal phosphate excretion via effects in the proximal tubule and decreasing intestinal phosphate absorption , 8. In pAt present, as with cholesterol in the early 1960’s, associative but not causal evidence exists relating serum FGF-23 to cardiac disease in humans, and little is known about the distribution of serum FGF-23 levels in patients who might be especially responsive to a medication aimed at lowering or blocking the pathophysiologic effects of FGF-23, such as those with CKD or congestive heart failure (CHF). Indeed, even research cohorts defined based on specific clinical characteristics  are generally not representative samples of the subgroups from which they are drawn. As a result, discretion is required when generalizing findings from these studies to more general populations.To bridge this gap, we use a predictive model generated using data from the FHS to impute serum C-terminal FGF-23 (cFGF-23) levels for adults in the US National Health and Nutrition Examination Survey (NHANES), and estimate the size of various patient subgroups with levels of cFGF-23 above selected thresholds. In addition, to examine whether a population using estimated FGF-23 values exhibits results congruent to studies with actual FGF-23 samples, we explore the association between estimated cFGF-23 levels (referred to throughout as eFGF-23) and mortality using the NHANES-provided linkage to mortality data from the National Death Index. Our goal is to provide a case study demonstrating the utility of this approach.This is a retrospective observational study using publicly available, de-identified patient-level data from the US NHANES. NHANES is an ongoing program of studies conducted by the National Center for Health Statistics and the Centers for Disease Control and Prevention to assess the health and nutritional status of adults and children in the US . NHANES 2) without albuminuria; stage 1, kidney damage with normal or increased GFR (≥90 mL/min/1.73 m2) and albuminuria; stage 2, kidney damage with mildly decreased GFR (60–89 mL/min/1.73 m2) and albuminuria; stage 3, moderately decreased GFR (30–59 mL/min/1.73 m2); stage 4, severely decreased GFR (15–29 mL/min/1.73 m2); stage 5, kidney failure  [We included all adult NHANES participants 1999–2014) aged ≥20 years in the study population. Subgroups of interest included participants with CKD, identified by laboratory measurements, and those with CHF, identified through patient report. Estimated glomerular filtration rate (GFR) and urinary albumin-creation ratio were used to determine CKD stage in accordance with the Kidney Disease: Improving Global Outcomes (KDIGO) 2012 clinical practice guideline for the evaluation and management of CKD: stage 0, mildly decreased or high GFR  . We used99–2014 aTo date, no NHANES cohort has included FGF-23. In order to estimate FGF-23, we used a regression equation from Haring and colleagues . This moHaring and colleagues provided an equation to estimate FGF-23 based on their Framingham subgroup, the median and interquartile range (IQR) for the FGF-23 distribution in this cohort, and the summary statistics of key demographic and clinical measures. However, the published equation does not include an intercept. Therefore, our approach was to include all of the information available to implement the equation, without an intercept, in an NHANES sub-sample that was similar to the Framingham sample, and then rescale to match the FHS sample’s FGF-23 summary statistics, hence estimating the intercept. The Haring FGF-23 equation utilizes standardized independent variables and standardized dependent variables of a natural log transformed FGF-23. In order to ensure that FGF-23 estimates in NHANES were on the same scale as the Framingham measurements, we identified a subgroup of NHANES subjects comparable to the Framingham cohort and calibrated estimates such that the distribution of eFGF-23 for this subgroup matched the observed distribution of FGF-23 in the Framingham cohort.To create this calibration subgroup, initially the NHANES age group of 43 years and above provided a subset with an age distribution similar to the Framingham cohort’s 58.8 (mean) and 11.2 (standard deviation). Next, the Framingham rate of 88.0% white was much higher than the rate of NHANES, so NHANES race and ethnicities were re-weighted to match the Framingham group. Lastly, even within this age and race/ethnicity matched subgroup, the published mean eGFR for Framingham subjects was lower than the NHANES mean. Differences in eGFR between NHANES and Framingham likely represent true differences in eGFR of the populations and not measurement variation. Therefore, only for the purposes of creating an FHS-type sample of NHANES, the NHANES subgroup’s eGFR values were “inflated” to have the same mean and standard deviation as those from Framingham. The resulting standardized ln(eFGF-23) was transformed so that the median and IQR was  within this subgroup of NHANES, equivalent to the FGF-23 results reported by Haring et al., hence addressing the problem of the missing intercept. For calculation of the NHANES estimates of eFGF-23, the actual eGFR measures were used, not the values transformed to match FHS.In order to understand the magnitude of differences between the source (FHS), target , and calibration cohorts, we examined the demographic and laboratory characteristics of these cohorts side-by-side. We also compared  the distribution of eFGF-23 levels for the NHANES cohort with the observed FGF-23 levels reported for the Framingham cohort. Distributions were also extended to subpopulations of interest within NHANES with various CKD stages, CHF, and combinations of both.sub); and (3) calculating the corresponding NNTs using the equation NNT = 1/ (MRsub−0.0325).In order to produce unbiased national estimates of the prevalence of elevated FGF-23 overall and within subgroups of interest, we utilized sample weights (in addition to strata and clusters for proper standard error estimation) from NHANES within SAS survey procedures. To assess the face validity of our eFGF-23 estimates, we used Kaplan-Meier estimates and Cox proportional-hazards regression models to examine the age- and sex-adjusted relationship between eFGF-23 and CV and all-cause mortality in NHANES and compared these estimates to those from comparable models reported by Haring et al. . FinallyAll statistical analyses were conducted using SAS 9.4 software .Subjects in the Framingham cohort tended to be older (mean age 58.8 vs. 46.2 years), less ethnically diverse (12% vs. 30.2% non-white), and had a higher prevalence of hypertension (45.7% vs. 30.1%), type 2 diabetes (11.2% vs. 7.3%) and CVD (12.4% vs. 8.1%) than the adult US population estimated from the target NHANES group . FramingThe eFGF-23 values from the overall weighted NHANES population were lower  than the only published FGF-23 percentiles from the Framingham cohort , most noth (47.4 RU/ml), 75th (64.0 RU/ml) and 90th (84.4 RU/ml) percentiles of the overall NHANES distribution, respectively. Similarly, nearly all (93.0%) NHANES subjects with stage 2 CKD have an eFGF-23 above the NHANES median, whereas 66.2% and 30.3% have values above the 75th and 90th percentiles. Among subjects with CHF, nearly all (96.2%) are above the NHANES median and about two-thirds (67.8%) have values above the 90th percentile.Using different thresholds for what constitutes an “elevated” FGF-23 level resulted in different population size estimates. For example, an estimated 79.1%, 46.6% and 19.7% of NHANES subjects aged 60 to 69 have eFGF-23 levels above the 50A different pattern emerges when considering which subgroups contribute most to the overall population of subjects with “elevated” eFGF-23 levels, as seen in The overall and CV mortality rates associated with eFGF-23 in the NHANES population are shown by quartile in The association between the continuous measure of eFGF-23 and estimated 10-year mortality was also significant in adjusted Cox proportional-hazards models . In our The potential population and individual level benefits from a hypothetical FGF-23-lowering agent are shown in In this study, we demonstrated an approach to using a published predictive model from Framingham to estimate FGF-23 values for all adult subjects in NHANES and, by applying NHANES sample weights, estimate the distribution of FGF-23 levels in the overall adult US population. The viability and utility of this approach depends on the degree of overlap between the source and target populations: a certain degree of overlap is required to support the projection. If too similar, estimation would be unnecessary, while insufficient overlap leads to excessive uncertainty due to extrapolation. In our case, the source population  was older and had a higher burden of CV disease than the target NHANES population, yet was comparable to a calibration cohort comprised of more than half of all adult subjects in NHANES. This situation may be more optimal relative to the typical situation in which early understanding of a novel biomarker is derived from smaller niche research cohorts with a disease process of interest. Establishing appropriate bounds on the use of this type of approach requires replication in scenarios with different degrees of source-target population overlap, and ultimately validation through direct measurement of biomarker values in epidemiologic cohorts such as NHANES.Perhaps the most salient finding relative to eFGF-23 from this study is the discrepancy between the groups with the highest values and those with the greatest number of subjects with elevated values. Mean eFGF-23 levels increased steadily with age and CKD, and were more than twice as high among those with versus without CHF. However, the subgroups with the largest absolute numbers of subjects in the top quartile of eFGF-23 were those with earlier stages of CKD and without CHF. This highlights a challenge faced in developing new therapeutics targeting FGF-23, particularly during late clinical development. While targeting patients from smaller subgroups with the greatest absolute risk  may increase the likelihood of technical success for the therapy, a strategy aimed at treating larger subgroups with greater overall population burden of disease may facilitate patient enrollment, permit market entry at a lower price, and increase the likelihood of commercial success. The absolute risk reduction would not be as high and thus the number needed to treat to prevent an adverse disease outcome would be higher relative to that in the smaller subgroup patients at greater risk. This tension between population and individual level treatment benefit is clearly apparent in the NNT estimates in The role of FGF-23 in disease has been extensively studied in the context of CKD. In CKD, the ability to balance the intake of dietary phosphate with the loss of phosphate through excretion by the kidney is impaired. The resultant increase in the body’s phosphate burden produces an increase in FGF-23 levels. The elevated FGF-23 may reflect tubular dysfunction and resistance to endocrine factors in kidney disease, an important pathology not fully captured by measurement of GFR or albuminuria .High levels of FGF-23 appear to have a causal role in CVD. Elevated FGF-23 is more closely associated to left ventricular hypertrophy and heart failure than atherosclerosis related CVD , 27, 28.At what concentration FGF-23 transitions from a bystander, risk associated factor, to a significant maladaptive, toxic, disease-causing factor in not clear . UnderstAmong this study’s other limitations is that certain variables included in the regression equation for FGF-23 were defined or characterized differently in Framingham versus NHANES, which may have introduced bias into our estimates. For example, age in NHANES is truncated at 85, placing a ceiling on the contribution of age to eFGF-23. In addition, the Framingham smoking definition reflects more than a cigarette per day over the past year, whereas NHANES utilized a recent 30-day time frame.Furthermore, in the absence of direct measurement of FGF-23 in at least a subset of NHANES patients, it is impossible to judge whether the Haring FGF-23 equation has external validity when applied to the more diverse NHANES population. The fact that the association between estimated NHANES FGF-23 values and CV and all-cause mortality was similar to what was observed by Haring and colleagues provides some support for the external validity of their equation. It is also interesting to note that the estimated FGF-23 levels for NHANES patients with CHF (not included in the Haring equation) were more than twice as high as among those without CHF, consistent with findings from other studies . NeverthA further limitation relates to the overlap in variables used to estimate FGF-23 and those used for risk adjustment in the mortality models. This could in part account for the decreased precision in the estimates of the associations between FGF-23 and CV and all-cause mortality in our models relative to what was observed by Haring and colleagues. Nevertheless, estimates from our models are directionally consistent with those from the Haring study, and most of the point estimates from Haring are contained within the confidence intervals from our study, as seen in Despite these limitations, we believe an estimate of the population distribution of biomarker values can inform research agendas and enhance drug research decision making. Data from analyses like these could be used to derive estimated numbers needed to treat (NNTs) to prevent a CV death among potential target subgroups at specific time intervals and assuming a variety of hypothetical efficacy levels . Researchers might then use insights gained not only to inform optimal target population selection but also to make value-based determinations of which programs to move forward or terminate.In conclusion, it is feasible to estimate the distribution of novel biomarker values in the general population by applying predictive equations from smaller research cohorts to weighted national survey data. The resulting information can provide insights into the relative size of subgroups with elevated values, informing future research efforts. With respect to FGF-23, whereas older subjects with advanced CKD and CHF have the highest estimated values, targeting larger subgroups with less advanced CKD and without CHF may lead to provision of more meaningful public health benefits. Ultimately, the optimal choice of treatment targets requires carefully weighing anticipated patient and population level benefits.


NCF-1 gene on chromosome 7. As remarkable facial features and psychomotor retardation are not associated with CGD, a more detailed genetic work-up using fluorescence in situ hybridization was performed. A microdeletion in 7q11.23 on one allele indicated Williams–Beuren syndrome (WBS). The NCF-1 gene and its two pseudogenes are part of a highly repetitive region within 7q11.23 and are prone to recombination events and deletions. Such deletions can involve both the WBS critical region and the NCF-1 wildtype gene, as was the case for our patient. The second allele of the NCF-1 gene was affected by the frequent c.75.76delGT mutation that stems from a recombination of the NCF-1 wildtype gene with one of its pseudogenes. In conclusion, patients with NCF-1-deficient CGD may also harbor microdeletions that result in WBS or other hereditary disorders; therefore, it is important to perform a thorough genetic analysis in order to initiate appropriate therapy for these patients.We report the case of a 19-years-old patient who presented with a perplexing variety of symptoms which included remarkable facial features, intellectual disability, granulomatous upper lip swelling , Crohn’s-like disease, non-productive cough, and a granulomatous mass localized in the left lung. Chronic granulomatous disease (CGD) was diagnosed using dihydrorhodamine 123 assay that showed low levels of phagocytic NADPH-oxidase. DNA sequencing revealed a heterozygous mutation in the  CYBA, NCF-1, and NCF-2 genes . Serum levels of soluble interleukin 2 receptor, neopterin, and angiotensin converting enzyme were negative. Bronchoalveolar lavage fluid showed lymphocytosis. Transbronchial biopsy revealed chronic, partly granulomatous inflammation with no signs of necrosis . No mycobacteria were found and the results were not typical for sarcoidosis. Steroid and antibiotic (cotrimoxazol) therapy resulted in rapid resolution of the intrathoracic mass.A 19-years-old patient was transferred to our pediatric hospital for further diagnostic analysis from a clinic specialized in adult lung diseases. The afebrile patient suffered from a dry, non-productive cough. An intrathoracic inflammatory granulomatous mass was localized by radiogram and computer tomography . No further assessment of the patient’s cognitive abilities had been performed; however, clinically a mild intellectual disability was observed. MRS (OMIM 155900) and Crohn’s disease (OMIM 266600) had previously been diagnosed. MRS was histologically diagnosed at the age of 10 when the patient presented with swelling of the upper lip and granulomatous inflammation. However, typical features, such as lingua plicata or facialis paresis were not noted . The patient had neither diarrhea nor significant gastrointestinal complaints. Colonoscopic biopsies revealed epithelial cell granulomas compatible with Crohn’s disease (see Table 1).The patient’s past medical history included the diagnosis of a learning disability . Phenotypical dysmorphic facies had been noted including broad forehead, bi-temporal narrowing, strabismus, long philtrum, wide mouth, and large ear lobes . The mutation was confirmed as heterozygous for the father, but surprisingly not for the mother. Unfortunately, symptomatic data for the father are unknown, as he does not reside with the family . The patient was administered the recommended prophylactic therapy of cotrimoxazol and itraconazol.A multisystem granulomatous disease was suspected; therefore, the patient was tested for CGD using a dihydrorhodamine 123 (DHR) assay. This assay revealed a considerable decrease in NADPH-oxidase activity. Subsequent Sanger sequencing identified the most common mutation within the in situ hybridization revealed microdeletions within 7q11.23 (OMIM 194050) with locus specific deletions; this confirmed the diagnosis of WBS.Nevertheless, neither learning disability nor facial dysmorphism are associated with CGD. The dysmorphism raised suspicion of WBS despite the absence of cardiac symptoms. Upon further genetic analysis, fluorescence NCF-1 gene analysis. The clinical course of our patient differed from most CGD patients, due to the fact that NCF-1- (p47phox-) deficient CGD is generally associated with residual NADPH activity. This may explain why our patient had no history of life-threatening infection even while under immunosuppressive therapy aimed at treating previously suspected MRS. However, although NCF-1-deficient CGD generally follows a milder course than CGD lacking residual activity, some infectious manifestations can be equally as severe and life threatening as those contracted during the course of classical CGD, e.g., invasive aspergillus infections of the lungs  with genetic variations have been identified. Each segmental duplication is composed of three differentiated blocks designated as “A,” “B,” and “C”   .NCF-1 gene. The other allele harbors the frequent GT-deletion in exon 2 (c.75.76delGT) of the NCF-1 wildtype gene. The mutation in NCF-1 occurs then in the heterozygous state. Note that the WBS is normally caused by a de novo microdeletion, whereas the NCF-1-deficient CGD is inherited in an autosomal recessive manner. We find it remarkable that our patient is afflicted by two separate disorders that share a common background: Gene duplication (and triplication) with genetic variation in the region 7q11.23 during evolution and more recently as a result of recombination events between these regions.These considerations apply to our patient in the following way: One allele of his 7q11.23 region harbors a large deletion of the critical WBS region that involves the NCF-1 gene increases the risk of hypertension in patients with WBS. It has also been found that the hemizygosity in NCF-1 gene decreases this risk (It has been reported that a functional his risk ; yet, mohis risk .NCF-1-deficient CGD. The identification of disease causing mutations using in-depth genetic analysis will facilitate the initiation of appropriate therapy for patients who are afflicted by these and other hereditary disorders.It is important to be aware of possible clinical indications suggestive of WBS in patients with Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the editor of this journal. The ethic committee approval has been obtained by institutional ethic committee of the medical faculty of the University Duisburg-Essen under file number 18-8226-BO and a copy of the approval is available for review by the editor of this journal.DS made substantial contributions to conception and design and involved in drafting the manuscript. AK and JR made substantial contributions to the acquisition, analysis, and interpretation of the data. LP, ME, and MO involved in critically revising the manuscript for important intellectual content. FS made substantial contributions to conception and design, acquisition, analysis, and interpretation of the data, involved in revising the manuscript critically for important intellectual content, and gave the final approval of the version to be published.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.


Nitrogen-vacancy color centers in diamond are a very promising medium for many sensing applications such as magnetometry and thermometry. In this work, we study nanodiamonds deposited from a suspension onto glass substrates. Fluorescence and optically detected magnetic resonance spectra recorded with the dried-out nanodiamond ensembles are presented and a suitable scheme for tracking the magnetic-field value using a continuous poly-crystalline spectrum is introduced. Lastly, we demonstrate a remote-sensing capability of the high-numerical-aperture imaging fiber bundle with nanodiamonds deposited on its end facet. Recent years have witnessed a substantial increase of interest in studies of color centers in diamond, in particular the nitrogen-vacancy centers. The negatively charged nitrogen-vacancy  center(>∼3 ms) , quantum(>∼3 ms) ,7, and q(>∼3 ms) . NV diam(>∼3 ms) .Individual NV centers allow for magnetic surface mapping with nanometric spatial resolution ,12,13,14Nanodiamonds (NDs) are generally obtained by detonation or crushing larger diamond samples. Their sizes cover wide range between single nano- to micrometers. They are available commercially with preselected sizes and other properties, such as customary surface functionalization. Additionally, substantial density of defects may be introduced to NDs by means of high-energy particle irradiation and subsequent annealing. For example, the ND fluorescence enhancement in the green or red wavelength region may be observed, caused by formation of nitrogen-vacancy-nitrogen (NVN or H3) and NV centers, respectively. Depending on particular applications NDs can be used as powders, suspensions in various liquids (possibly with microfluidic devices), and single NDs deposited in a given location or manipulated with optical tweezers ,17,18,19Many ND applications, such as seeding of substrates for deposition of thin diamond layers via the chemical vapor deposition or preparation of ND-doped polymer nanocomposites and galvanic coatings, require their deposition in a liquid form, hence NDs are often dispersed to form colloidal suspensions . Good unIn this work, we present the results of our research on the use of ND powders, which offer an interesting trade-off between sensitivity, resolution and manufacturing costs. We demonstrate the sensing potential of two platforms using NDs with NV centers, i.e., planar layers of dried suspensions and an ND-coated imaging fiber bundle (IFB).The experimental system is constructed around a confocal fluorescence microscope shown schematically in For optically detected magnetic resonance (ODMR) measurements, a microwave signal from a generator (SRS SG396) is fed through a high-power amplifier  and delivered to a loop-gap type antenna structure on a printed circuit board (PCB). The structure is similar to the one described in Reference  and is dWe use two types of ND solutions. The first one is a commercially available 140-nm-size ND suspension in de-ionized water . The NDs have a high ∼2–3 ppm NV concentration and their surface is carboxylated to stabilize the suspension. The other suspension is made in-house from a  be used .The first type of samples we study is formed by depositing droplets of ND solution on a glass substrate, typically a microscope slide or a coverslip  and shoWhile in zero magnetic field the measured NDs ODMR spectrum did not differ significantly from the single-crystalline one, in non-zero field there are significant differences. The spectra are strongly inhomogeneously broadened by the random orientation of nanocrystals with respect to the direction of the magnetic field ,31. ThisThe outermost edges of the spectra correspond to the NDs with the maximal projections of the magnetic field, i.e., those oriented along the field direction. Approximately linear increase of the spectrum width with the field strength is visible in Magnetometry using single-crystal NV diamonds is already well established . One comIn non-uniform samples, such as formed by dried ND suspensions, a spatially varying fluorescence level strongly obstructs the recording of small (∼0.1–1%) ODMR contrast changes between various points on the sample. Additionally, with a poor thermal contact, the ND ensemble can have a wide distribution of temperatures making the analysis of recorded signals even more difficult. One of the ways to alleviate these limitations is the application of amplitude or frequency modulation techniques for the microwave-field and a simultaneous phase-sensitive (lock-in) detection ,32,33. SS is the normalized fluorescence signal, and In order to assess the DC magnetic-field sensitivity, we follow the analysis presented in Reference . We assugiven by (1)η=δBmUsing the Zeeman shift formula, signal slope value of For practical application of ND layers, we propose a sensing architecture consisting of a high numerical-aperture imaging fiber bundle with one of the facets ND-coated. In a proof-of-concept experiment, we have used a short (length of ~12 mm) section of an IFB. The IFB was fabricated identically to the FB 1–3 samples presented in Reference , except One end of IFB was coated by depositing a droplet of the 1-A central feature of the IFB is that it enables simultaneous observation of the ND fluorescence over the entire distal-end surface. We have recorded a fluorescence map by scanning the IFB under the microscope and it is shown in Since the IFB acts in our experiments as a relay of the optical fluorescence signal, its most important parameter is the high numerical aperture, NA In summary, we have reported on experiments with dried ND suspensions aiming at the development of photonic sensors with imaging feasibility. With preselected ND sizes and two different solvents we worked towards preparation of future microfluidic applications and/or experiments with optical tweezers with liquid biological samples. In addition to determining static parameters of the samples, we expect the ND ODMR methodology to be helpful in studies of the dynamics of various bio-samples and their constituents moving in a liquid phase.We deposited dry ND layers containing NV centers on glass structures and found conditions responsible for their various spatial distributions. After illumination with green laser light these layers emitted strong red fluorescence which was recorded with good signal-to-noise ratio by a regular avalanche photodiode detector. Moreover, with proper MW excitation and optical detection, we were able to record magnetic resonance spectra characteristic for the local magnetic field. ODMR spectra of a deposited layer in non-zero magnetic fields exhibit substantial inhomogeneous broadening caused by random orientations of NV spins. While, generally, such broadening reduces magnetic sensitivity, a careful analysis of the broadened spectra may enable accurate determination of the magnetic-field intensity and open the way to magnetic imaging and mapping. In addition, sensing of other modalities, such as temperature and strain, may be possible with NDs .Along with measurements of ODMR signals in NDs deposited on glass, we have also coated the imaging optical fiber bundle . In addiIn conclusion, several described experiments illustrate the suitability of ND ensembles with high NV concentration for precision magnetic-field sensing. They also demonstrate the significant potential of ND layers as a main part of various photonic imaging sensors.


Immunooncology is still a growing area in cancer therapy. Drugs within this therapeutic approach do not directly target/attack the tumor but interfere with immune checkpoints and target or reprogram key metabolic pathways critical for anti-cancer immune defense. Indolamine 2,3-dioxygenase 1 (IDO1) and the tryptophan (TRP)-kynurenine pathway were identified as critical mechanisms in cancer immune escape and their inhibition as an approach with promising therapeutic potential. Particularly, a multitude of IDO1 inhibiting tryptophan analogs are widely applied in several clinical trials. However, this therapy results in a variety of implications for the patient's physiology. This is not only due to the inhibition of an enzyme important in almost every organ and tissue in the body but also because of the general nature of the inhibitor as an analog of a proteinogenic amino acid as well as the initiation of cellular detoxification known to affect inflammatory pathways. In this review we provide a deeper insight into the physiological consequences of an IDO1 inhibiting therapy based on TRP related molecules. We discuss potential side and off-target effects that contribute to the interpretation of unexpected positive as well as negative results of ongoing or discontinued clinical studies while we also highlight the potential of these inhibitors independent of the IDO1 signaling pathway. The degradation of tryptophan (TRP) along the kynurenine pathway (KP) plays a crucial role in the regulation of the immune response, notably as a counter-regulatory mechanism in the context of inflammation , 2. Threin vitro as well as in clinical trials . The application of IDO inhibitors should prevent both the depletion of TRP and the production of immunomodulatory TRP metabolites such as kynurenine (KYN) or kynurenic acid (KYNA) contributing to a suppression of IDO-induced immune escape of cancer cells. Although some promising results were described in vitro and in rodent models  of ~7 μM  and 1-methyl-L-tryptophan (L-1-MT) were scrutinized as IDO inhibitors. Indoximod, which is under investigation in several clinical trials, showed therapeutic effects in murine tumor models reversing the suppression of T-cell proliferation and inducing retardation, but no total arrest of tumor growth , 10, 13.in vivo. A phase I trial of tumor patients using Indoximod as an IDO1 inhibitor has shown that doses higher than 1,200 mg 1-MT in a patient do not increase peak serum levels over ~16 μM (50) of L-1-MT and D-1-MT was 120 μM and more than 2.5 mM, respectively. As shown for 1-MT, it should be considered that Ki values from cell-free assays may not reflect the inhibitory effect in vivo resulting in inefficient inhibition at physiological concentrations. According to the reported IC50 values in HeLa cells it is assumed that both L- and D-1-MT are in vivo relatively ineffective IDO inhibitors. Nevertheless, significant effects of these drugs on immune response and KP reveal off-target modes of actions. The ineffective IDO inhibition by 1-MT in vivo should be considered for the interpretation of published studies using 1-MT.One reason for the lack of effective IDO1 inhibition could be that the concentration of 1-MT was too low to inhibit IDO1 activity r ~16 μM , indicatr ~16 μM , increasr ~16 μM . Interesr ~16 μM . This in50 = 1.5 μM) selectivity against the later enzyme (50 inhibition of TDO. This may explain some of the effects and side effects of the treatment. The question to what extent a combined IDO1 and TDO inhibition is advantageous or disadvantageous is currently still under debate. A recently published article demonstrates the quickly absorption and moderate bioavailability of Navoximod and shows that this drug is extensively metabolized mainly by UDP-glucuronosyltransferases (UGT)  . It woulIn vitro studies reveal that Epacadostat decreases the proliferation of regulatory T-cells concurrent with an increase of activity of cytotoxic T-lymphocytes . Preliminary results of a clinical phase 1/2a trail with BMS-986205 alone or in combination with Nivolumab demonstrate that an IC90 can be reliably achieved with 200 mg daily oral application with this IDO inhibitor , Epacadostat, Navoximod, and Norharmane activate the ubiquitously expressed promiscuous ligand-operated arylhydrocarbon receptor (AhR) , 40. ThiThe activation of AhR by IDO inhibitors may be detrimental to the assumed treatment concept of downregulating the KP by IDO inhibition. This supports several studies describing pro-carcinogenic effects of AhR ligands in several human cancers including prostate, lung, breast, pancreatic and gastric cancer . In tranAn important off-target mode of action of TRP-related IDO inhibitors is that they mimic TRP even in the context as fake nutritional signals. They may target the mammalian target of rapamycin (mTOR) signaling which is the central pathway in amino acid sensing and signaling . Activatin vitro that the 1-MT-induced increase in TRP may impede the antimicrobial and immunoregulatory functions of LPS-induced TRP depletion (It has been shown epletion , facilitepletion . During epletion . Tryptopepletion . Most TRepletion . Howeverepletion . This main vitro as well as in clinical trials. However, there are controversial results regarding the efficacy of IDO inhibitors for IDO inhibition and cancer treatment. It should be considered that some competitive inhibitors such as 1-MT or Norharmane can hardly induce effective in vivo IDO inhibition due to their low in vitro potency and the fact that they reach plasma concentrations similar to those of the IDO substrate TRP. Therefore, the immunomodulatory effect of these inhibitors in the body is most likely related to off-target effects such as AhR activation or fake nutritional signaling rather than IDO inhibition. This should be considered when interpreting the significance of IDO in in vitro and in vivo studies with these inhibitors. In contrast to 1-MT and Norharmane, more potent inhibitors such as Epacadostat or Navoximod inhibit IDO on nanomolar levels resulting in decreased KYN production in vitro and in vivo. Nevertheless, the failure of a large phase III clinical trial with Epacadostat in melanoma patients indicates that IDO, at least in this type of cancer, is not a suitable target to improve the efficacy of drugs such as Pembrolizumab. In this context, it might be beneficial, for example, to use a molecular diagnostic approach such as biomarker profiles to clarify in advance to what extent IDO inhibition can be beneficial for the respective cancer treatment strategy. Furthermore, the pro- and anti-carcinogenic effects of mTOR and/or AhR activation may be a double-edged sword depending on the type of cancer, the tumor environment and the concurrent anticancer treatment. These side effects, which have so far not been considered sufficiently, should definitely be taken into account in the development of beneficial synergistic drug combinations in cancer therapy. In this context, computational approaches and bioinformatic modeling could become increasingly important in the future.Several IDO1-inhibitors are currently tested EW and JG wrote the first draft of the manuscript. JD critically revised the manuscript for important intellectual content. Each author has approved the final version of the manuscript before publication.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.


Obesity is associated with a higher risk of developing breast cancer and with worse disease outcomes for women of all ages. The composition, density, and organization of the breast tissue stroma are also known to play an important role in the development and progression of the disease. However, the connections between obesity and stromal remodeling are not well understood. We sought to characterize detailed organization features of the collagen matrix within healthy and cancerous breast tissues acquired from mice exposed to either a normal or high fat (obesity inducing) diet. We performed second-harmonic generation and spectral two-photon excited fluorescence imaging, and we extracted the level of collagen-associated fluorescence (CAF) along with metrics of collagen content, three-dimensional, and two-dimensional organization. There were significant differences in the CAF intensity and overall collagen organization between normal and tumor tissues; however, obesity-enhanced changes in these metrics, especially when three-dimensional organization metrics were considered. Thus, our studies indicate that obesity impacts significantly collagen organization and structure and the related pathways of communication may be important future therapeutic targets. We acquired 3-D SHG image stacks at 920-nm excitation/460-nm emission and 2-D two-photon excited fluorescence (TPEF) spectral images at 740- and 860-nm excitation and 390- to 750-nm emission. We analyzed these images using automated algorithms to extract quantitative metrics of 3-D fiber alignment and content, 2-D collagen texture features, and CAF. We report on the presence of significant differences in several of these metrics between normal and tumor tissues; notably, differences in 3-D alignment and CAF are enhanced in the context of obesity.22.1ad libitum. To generate tumors, Animal procedures were performed in accordance with a protocol approved by the University of Wisconsin–Madison Institutional Animal Care and Use Committee. Mice were housed and handled in AAALAC-accredited facilities in accordance with guidelines from the National Institutes of Health (NIH) Guide for Care and Use of Laboratory Animals. All female mice were purchased from Jackson Laboratories . In this study, twenty 8-week-old female C57Bl/6 mice were fed either a control  diet  .2.3.3SHG-positive pixels were considered any pixels with nonzero intensity values following integration of images collected at 420, 430, and 440 nm emissions acquired at 860 nm excitation. The corresponding mask for each field was applied to all emission sequences acquired at 740- and 860-nm excitation wavelengths. To quantify the fluorescence signal emanating from the SHG-positive pixels, we integrated all the corresponding pixel intensities within each emission band to generate a spectrum from a given imaged field. The intensity of each spectrum was normalized by the intensity of the SHG signal peak value (detected at 860-nm excitation/430-nm emission) for both wavelengths. The alternating least square algorithm provided in the Matlab PLS toolbox  was used to deconvolve the spectra into the spectral shapes and corresponding weights of two components.2.4Hematoxylin and eosin (H&E) and anti-2.5All image processing was performed in MATLAB_R2018a software. Statistical analysis was performed in JMP Pro 14 . Significance in the difference of the values of the different metrics among different groups was calculated based on one-way ANOVA and post-hoc Tukey tests. Comparisons were performed to assess differences either as a result of the presence of tumor or in response to diet . Significant differences at 33.1Supplementary Material.Representative SHG stacks . The decrease in sensitivity of the 3-D variance to report on differences between some of the groups as the window size increases reflects the presence of heterogeneities in alignment that are characterized more accurately with smaller window sizes. The averaged PDFs of the localized 3-D variance values of each sample within a group are shown in Supplementary Material. Within the normal tissues, we observe that fields that include a duct or a significant number of adipocytes tend to have broader and/or low-value shifted PDFs. While it is harder to observe variations in the PDF shapes of the tumor tissues that depend on a somewhat consistent fashion on the level of cellularity or adipocity, it is clear that these PDFs are shifted to lower 3-D variance values when compared to the normal tissues, and we see such shifts more consistently for the HFD-fed tumor-bearing mice. The KLD values reported in The changes in alignment are quantified first using the 3-D stacks and the corresponding 3-D directional variance metrics , calcula3.2Supplementary Material], and the tumor weights in HFD-fed mice were significantly larger than those from ND-fed mice [Fig. S4(B) in the Supplementary Material], the tumors appeared similar for the different diet-treatment groups using H&E staining [Figs. S4(C)–S4(D) in the Supplementary Material], as well as the expression of positive cancer-associated fibroblasts via immunohistochemical Supplementary Material]. Thus, these traditional 2-D staining procedures did not suggest differences in extracellular matrix remodeling. To further characterize 2-D SHG image texture, we used a GLCM-based approach. Representative SHG optical sections, along with corresponding cloned SHG images are shown in While the HFD-fed mice in our studies gained significantly more weight than those fed the ND [Fig. S4(A) in the 3.3,Average fluorescence emission spectra from each group of breast tissues we considered are shown from the SHG-positive pixels for 740- and 860-nm excitation in 4Our goal was to assess the impacts of obesity on metrics of collagen organization and structure within the matrix of normal and tumor breast tissues, as assessed by quantitative analysis of high-resolution SHG and TPEF spectroscopic images. We identified metrics of 3-D orientation/alignment , 2-D organization (,,,,–The increase in the levels of collagen fiber alignment  and CAF Statistically significant increases in fiber alignment and CAF are further enhanced within tumors excised from obese mice fed an HFD compared to tumors developed in mice fed a normal diet . Our fin,–In principle, obesity and diabetes have been associated with matrix stiffening that may be at least partially mediated by AGE cross-links.,Our evaluations of 3-D and 2-D collagen fiber morphology suggest that metrics that are sensitive to the 3-D orientation of the fibers are more sensitive than ones that exploit only 2-D image features. This finding is consistent with previous studies in which we performed more direct comparisons of the orientation variance of the fibers in 2-D and 3-D contexts and showed that the 3-D-based analysis was a more sensitive reporter of subtle changes.in vivo either through probes or minimally invasive procedures that may expose the tissue would overcome these limitations. Finally, as with any high-resolution imaging technology that samples a limited region of the tissue, there are always concerns that the regions selected for sampling may result in biased assessments. To avoid introducing any potential bias in our calculations, we made an effort to sample the diversity of features present in every sample visualized following a 2-D tile scan of a large (For our studies, we examined breast tissues that had been flash frozen immediately upon excision from mice. Obesity was induced in mice through the introduction of an HFD, and tumors were induced through the implantation of EO771 cells, which were derived from a spontaneous adenocarcinoma from a C57Bl/6 mouse. These tumors grow faster than typical transgenic models of breast cancer and are not typically associated with a significant stromal component, which is consistent with the overall decrease in fiber density that we observe within the tumor tissues. However, it will be important to extend our studies to different breast cancer models to determine whether we observe similar trends in terms of the impacts of obesity. Also, some degradation, especially in the CAF signal, and structural changes are possible upon freezing and thawing of the tissue. Since, we followed identical tissue handling protocols for all samples, we anticipate that, even if present, such processing-induced changes would impact in a similar way all our samples and would not affect our main results that focus on matrix differences between groups. In addition, previous studies performed with frozen-thawed chick embryo tendon tissues, indicated a correlation of CAF intensity with the levels of LOX-mediated crosslinks assessed by mass spectrometry.In summary, our study demonstrates the potential of label-free two-photon imaging to assess complementary features of collagen fiber organization and structure and to provide useful and highly quantitative insights regarding the remodeling processes that occur in breast cancer. Exploitation of the 3-D nature of the images we acquire using SHG imaging enhances our sensitivity to potentially subtle changes that may remain undetected in some cases if the analysis is limited to 2-D images. Acquisition of SHG and TPEF images simultaneously is possible and can provide information regarding multiple aspects of collagen organization and structure, all of which may ultimately play an important role in affecting cell behavior. Thus, their combination could be a powerful tool in helping us to understand better the complex role of risk factors, such as obesity, in the development and progression of breast cancer.Click here for additional data file.


Delayed diagnosis and treatment initiation of smear-negative tuberculosis (TB) patients can lead to increased morbidity and mortality, particularly among those co-infected with the human immunodeficiency virus (HIV).® MTB/RIF testing at five rural tertiary hospitals in Uganda.To compare TB treatment initiation among smear-negative presumptive TB patients in the 6 months before and after the introduction of Xpert2 tests. Time to treatment was analysed using Kaplan-Meier survival analysis. Patient records of the dates and results of sputum analysis were extracted from TB laboratory registers and linked to those on treatment initiation as indicated in the TB treatment registers. The proportion of smear-negative presumptive patients who initiated anti-tuberculosis treatment was compared before and after Xpert implementation using χP < 0.01). However, 37% (32/87) of patients with a confirmed TB diagnosis did not initiate treatment. Time to TB treatment initiation improved from 8 to 3.5 days between the study periods.Records from 3658 patients were analysed, 1894 before and 1764 after the introduction of Xpert testing. After the introduction of Xpert, 25% (437/1764) of smear-negative presumptive TB patients underwent testing. The proportion initiated on anti-tuberculosis treatment increased from 5.9% (112/1894) to 10.8% (190/1764) (Xpert testing was associated with improved TB treatment initiation among smear-negative presumptive TB patients. Improved utilisation and linkage to treatment could improve the impact of this test on patient-centred outcomes. TUBERCULOSIS (TB) REMAINS A MAJOR cause of morbidity and mortality among patients with the human immunodeficiency virus (HIV), even in the era of antiretroviral therapy (ART). In 2015, an estimated 1.2 million people who developed TB worldwide were co-infected with HIV and, among HIV-TB co-infected individuals, mortality was more than double that among TB patients without HIV.8® MTB/RIF assay , an automated molecular test with diagnostic sensitivity and specificity comparable with sputum culture,In 2011, the XpertWe aimed to assess the effect of the introduction of Xpert on the proportion of smear-negative presumptive TB patients initiated on anti-tuberculosis treatment and on the time to treatment initiation in these patients at selected tertiary hospitals in Uganda. We hypothesised that a more sensitive, rapid tool for TB confirmation would increase the proportion of smear-negative presumptive patients (the majority of whom are HIV-infected) treated for TB and reduce the time to treatment initiation among these patients.RRHs are tertiary hospitals that, in addition to in-and out-patient care, offer specialist clinical, radiological and surgical services. Each hospital serves 10 districts, a population of about 2 500 000, and has medical out-patient clinics which see 7000–10 000 patients each month. An HIV/ART clinic attached to each medical out-patient clinic serves 3000–5000 active TB patients.The results of all sputum samples tested in these laboratories were recorded in the hospitals' TB laboratory registers. TB treatment is offered at all RRHs using internationally recommended fixed-dose regimens provided by the Uganda NTLP, and is recorded in the hospitals' TB treatment register.From January 2012 to December 2014, Xpert testing was sequentially introduced at all RRHs in Uganda. Five of these hospitals were included in the study based on the availability and completeness of data 6 months before and 6 months after the introduction of Xpert. These hospitals serve large rural populations in Eastern, Northern and Western Uganda. Before the introduction of Xpert, presumptive TB patients with a smear-negative result were started on anti-tuberculosis treatment if they 1) had a chest X-ray suggestive of TB, 2) did not respond to a 2-week course of antibiotics, or 3) if the health care worker made a clinical decision to start them on anti-tuberculosis treatment . After tPatient records were retrieved from the hospital's TB laboratory register 6 months before and 6 months after the introduction of Xpert. Records from presumptive TB patients who were aged ⩾15 years, had at least one negative smear result and were HIV-positive or had unknown HIV status were included in our study. Records from patients whose HIV status was recorded as negative were not included in the study because, for the period covered by this analysis, Xpert was not recommended for HIV-negative patients. Records were excluded if patients' second smear result was positive or if they were diagnosed with RMP-resistant TB. Data on dates of sputum examination, dates of Xpert testing and the results of these tests were recorded. The data were then linked to results from the hospital TB treatment registers using a manual linkage system based on four parameters: the patient's name, age and sex and place of residence. From the hospital treatment registers, data on the date of TB treatment initiation, type of TB regimen and ART initiation (if the patient was co-infected with HIV) were extracted. For patients diagnosed at the tertiary hospital but started on treatment at a lower-level health facility, data on treatment initiation were obtained from the district TB register.For this study, a presumptive TB patient was described as a patient with two or more (one or more if HIV-positive) of the following TB symptoms: 1) cough for >2 weeks (cough of any duration in those who were HIV-positive), 2) persistent fevers, 3) night sweats, or 4) unexplained weight loss, as described in the WHO intensified case finding guide.Patients were initiated on treatment if they had anti-tuberculosis treatment with an internationally recommended, fixed-dose TB regimen that was recorded in the hospital's TB treatment register.Time to treatment initiation was defined as the number of days between the first smear test result and the start of anti-tuberculosis treatment.2 tests. The time to TB treatment initiation for the period before and after Xpert implementation was analysed using Kaplan-Meier survival analysis curves. The survival distributions of the two time periods were compared using the log-rank test.Univariate analyses of the characteristics of the patients seen at the hospitals and those undergoing Xpert were described using frequencies and percentages. Multivariate logistic regression was used to identify the patient characteristics associated with undergoing Xpert. The proportion of smear-negative presumptive patients initiated on treatment was compared for the period before and after Xpert implementation using χThe study protocol was approved by the Mengo Hospital Research and Ethics Review Board, Kampala (787/10–15), and by the Uganda National Council of Science and Technology, Kampala, Uganda (HS 1998).n = 69) or their HIV status was recorded as negative (n = 611). Records from 3658 patients were analysed: 1894 patients before and 1764 patients after the introduction of Xpert testing. Of these, 302 were initiated on anti-tuberculosis treatment: 5.9% (112/1894) patients before and 10.8% (190/1764) patients after the introduction of Xpert testing  after. However, the proportion of presumptive patients initiated on anti-tuberculosis treatment was lower than the proportion with a confirmed TB diagnosis, due to inadequate linkage to treatment of patients diagnosed with TB (The proportion of smear-negative presumptive TB patients initiated on anti-tuberculosis treatment increased from 5.9% (112/1894) before the introduction of Xpert to 10.8% (190/1764) ( with TB . Overall with TB .Anti-tuberculosis treatment was initiated more rapidly among patients with smear-negative TB after the introduction of Xpert. The mean time to treatment was reduced from 8 days before the introduction of Xpert to 3.5 days after. Survival analysis curves showed a statistically significant reduction in time to TB treatment initiation after the introduction of Xpert .This retrospective study showed low utilisation of Xpert in the initial months of implementation, with only 25% of all eligible patients undergoing Xpert. Low utilisation of Xpert in the initial months of implementation has been demonstrated by another study from Uganda, in which only 21% of all smear-negative presumptive TB patients underwent Xpert,In our study, the implementation of Xpert was associated with an increase in TB treatment initiation among smear-negative presumptive TB patients. These findings are similar to observations from studies carried out at tertiary hospitals in Uganda and India, where the introduction of Xpert increased the proportion of smear-negative presumptive TB patients started on treatment by respectively 10% and 28%.The impact of Xpert on the number of patients initiated on treatment was weakened by inadequate linkage to anti-tuberculosis treatment for patients diagnosed with TB: 37% of Xpert-positive patients were not initiated on anti-tuberculosis treatment. This scenario has been reported in studies from other high-burden countries such as South Africa and Mozambique, where respectively 24% and 33% of patients diagnosed with TB using Xpert testing were not started on treatment.Similar to findings from South Africa and Brazil, Xpert was associated with reduced time to TB treatment initiation.29Our study had two main limitations. First, as this is a quasi-experimental pre-post study, the observed increase in TB treatment initiation could be attributed to other improvements in health care delivery, such as the training of health care workers or improved record-keeping between the two time periods. This limitation was minimised by keeping the period of analysis short. Second, the study data collected under routine programmatic conditions resulted in the exclusion of health facilities with incomplete patient records, which may have introduced bias into the study. However, despite these limitations, the study has shown the impact of the benefits of Xpert in reducing treatment initiation delays among smear-negative patients and highlighted improvements that could be made in health care delivery to achieve greater benefits from this test.Xpert testing was associated with improvements in TB treatment initiation among smear-negative presumptive TB patients. Suboptimal utilisation and inadequate linkage to anti-tuberculosis treatment reduced the impact of Xpert testing. Health systems strengthening approaches that focus on improving utilisation and linkage to treatment for patients diagnosed with TB could improve the impact of this test on patient-centred outcomes.


Although animal and human studies have demonstrated interactions between dietary choline and fetal alcohol spectrum disorders, dietary choline deficiency in pregnancy is common in the US and worldwide. We sought to develop and validate a quantitative food frequency questionnaire (QFFQ) to estimate usual daily choline intake in pregnant mothers.A panel of nutrition experts developed a Choline-QFFQ food item list, including sources with high choline content and the most commonly consumed choline-containing foods in the target population. A data base for choline content of each item was compiled. For reliability and validity testing in a prospective longitudinal cohort, 123 heavy drinking Cape Coloured pregnant women and 83 abstaining/light-drinking controls were recruited at their first antenatal clinic visit. At 3 prenatal study visits, each gravida was interviewed about alcohol, smoking, and drug use, and administered a 24-hour recall interview and the Choline-QFFQ.Across all visits and assessments, > 78% of heavy drinkers and controls reported choline intake below the Dietary Reference Intakes adequate intake level (450 mg/day). Women reported a decrease in choline intake over time on the QFFQ. Reliability of the QFFQ across visits was good-to-acceptable for 2 of 4 group-level tests and 4 of 5 individual-level tests for both drinkers and controls. When compared with 24-hr recall data, validity of the QFFQ was good-to-acceptable for 3 of 4 individual-level tests and 3 of 5 group-level tests. For controls, validity was good-to-acceptable for all 4 individual-level tests and all 5 group-level tests.To our knowledge, this is the first quantitative choline food frequency screening questionnaire to be developed and validated for use with both heavy and non-drinking pregnant women and the first to be used in the Cape Coloured community in South Africa. Given the high prevalence of inadequate choline intake and the growing evidence that maternal choline supplementation can mitigate some of the adverse effects of prenatal alcohol exposure, this tool may be useful for both research and future clinical outreach programs. Fetal alcohol spectrum disorders (FASD) comprise a continuum of alcohol-related neurodevelopmental disorders ranging from the most severely affected children with fetal alcohol syndrome (FAS) to nonsyndromal children who also exhibit neurocognitive and/or behavioral deficits but may lack the facial features or growth deficits seen with FAS , 2. WorlA growing body of studies in FASD animal models has demonstrated that optimal maternal choline status can mitigate some of the teratogenic effects of alcohol , 10–16. rd-trimester dietary regimens – 480 mg choline/day  vs. 9300 mg/day .We have recently demonstrated that, although the diet and body composition of heavy drinking pregnant women is similar to that of abstaining/light-drinking pregnant women in socio-economically disadvantaged areas in Cape Town, South Africa, in repeated 24-hr dietary recall interviews, almost 90% of the women reported inadequate intake of choline . High raWe developed a quantitative choline food frequency questionnaire (Choline-QFFQ) to estimate usual dietary choline intake (mg/day) by participants in our Cape Town randomized clinical trial conducted to assess feasibility and efficacy of a maternal choline supplementation intervention conducted with heavy drinking women during pregnancy , 15. TheThe Choline-QFFQ food item list was developed by a panel of three nutrition experts in the Division of Human Nutrition, University of Cape Town, including M.S, S.B, and B.N., in which food sources with high choline content, as well as the most commonly consumed foods in the target population were included. The food sources list was derived from Nel and Steyn  and multThe picture sort method was used for administration of the Choline-QFFQ, whereby participants were asked to sort picture cards of food items included in the food list into two piles, those consumed during the past week and those not consumed during the past week. Interviewers, either a registered dietician or a research assistant with extensive training in dietary interviewing by M.S., then proceeded to quantify the frequency of intake and typical portion size for items that had been consumed during the past week. Portion size estimation was conducted using pictures and portion size props Fig.  includedM = 21.1 weeks gestation, SD = 6.1) regarding her alcohol consumption both at time of conception and recruitment and again at 4 and 12 weeks after recruitment, by a research nurse using the timeline follow-back interview  community who were recruited to participate in the choline clinical trial , 15 or tw  adaptedw  and reference method  were administered at each visit, with the reference method given first. The reliability of the Choline-QFFQ was tested by comparing the results of three administrations of the questionnaire. The validity of the Choline-QFFQ was tested using a dietary reference method . 24-hr dietary recall interviews were administered using the multiple-pass method  and pict® does not provide values for choline content, a database was created to estimate the choline content of foods consumed by the women in the study. All foods reported by women in 24-hr dietary recall interviews and any additional foods included in the Choline-QFFQ  were matched to preparation-specific food items in the USDA Database for the Choline Content of Common Foods (Release 2 [Since FoodFinder3elease 2 ). Cholinelease 2 . Inadequelease 2 .https://www.hsph.harvard.edu/donna-spiegelman/software/icc9/).Statistical analyses were performed using SPSS v.23 software . Reliability and validity analyses were conducted separately for drinkers and abstainers/light-drinkers. Reliability of the Choline-QFFQ was assessed by comparing the daily dietary choline intake values between visits 1 and 2; visits 2 and 3; and visits 1 and 3, adapting the validity analyses and interpretations suggested by Lombard et al. . ValiditThe majority of the mothers (89.8%) were 20-40 years of age, and drinkers were 2 years older than abstainers/light-drinkers on average Table . Three-fA large majority of women in both the heavy drinking and abstaining/light-drinking groups reported choline intake below the AI Table . Both heWhen examining the group-level reliability of the Choline-QFFQ among heavy drinkers between visits, results for percentage difference pointed to good agreement, while agreement reflected by the Wilcoxon signed rank test was poor Table . In BlanAmong drinkers, agreement was good-to-acceptable on most group-level tests, with the exception of percentage difference between QFFQ values at visit 1 and the mean of the 24-hr recall interviews (reference method) and the LOA index when averaging QFFQ values from visits 1-2  and resources (food identification cards and portion estimation props) to administer. To our knowledge, this is the first Choline-QFFQ to be developed and validated for use with pregnant women and the first to be used in the Cape Coloured community in South Africa. While this choline quantitative FFQ presented is specific to the Cape Coloured population, the development and validation steps we employed may be used to develop population-specific choline quantitative screening FFQs in other populations. Furthermore, the Choline-QFFQ performed well in our study population, suggesting that a Choline-QFFQ  can be used even in populations in which education is poor and/or socioeconomic status low. Despite extensive prevention efforts and guidelines ACOG, 2011) and 4 decades of scientific research demonstrating teratogenic effects of prenatal alcohol exposure, women worldwide continue to drink during pregnancy. FASD comprise the most common preventable cause of neurodevelopmental disabilities, with prevalence estimates of 1.1-5.0% in the US and Western Europe , 41 and  and 4 de minutes The steps of development and validation we employed can be used to develop population-specific choline quantitative screening FFQs in other populations. Development and validation of a nutrient-specific screening QFFQ involved several steps, including 1) development of the local food list; 2) determining the most appropriate recall period, frequency options and portion size estimation method(s), based on the aim of the dietary intake assessment, the target population, and time available for the administration; 3) identifying an appropriate reference method; and 4) identification of interpretable statistical tests for examining the reliability and validity of the newly developed QFFQ. The food list for our Choline-QFFQ was developed based on the integration of the best known dietary sources of choline and knowledge of commonly consumed foods in the Cape Coloured community in Cape Town by a panel of nutrition researchers with expertise on the dietary patterns of the target population. The recall period was set at the previous week based on recommendations by Willett . PortionWe used the statistical tests and interpretations recommended by Lombard et al.  to assesReliability and validity are measured separately, but results of the tests should be interpreted in light of each other. Although the QFFQ demonstrated both reliability and validity, tests of validity were more consistently acceptable-to-good than tests of reliability, particularly at the group level, as reflected in the Wilcoxon and Bland Altman bias tests, and the individual-level, when examining agreement as to whether a woman had inadequate intake. The relatively poorer reliability seen in these tests is likely due to the fact that women reported lower choline intake at each visit. This trend may reflect interview fatigue, as has been reported in several other studies ; after fThis study had limitations common to other longitudinal studies of nutrition. 24-hr dietary recall interviews can yield inaccurate estimates of usual intake due to recall errors. However, we have previously reported that intraclass correlations and within-subject coefficients of variation for dietary nutrient intakes in this cohort, including choline, were similar to those of NHANES and other peer-reviewed epidemiologic studies in the U.S. , indicatTo our knowledge, this is the first quantitative choline food frequency screening questionnaire to be developed and validated for use in both heavy and non-drinking pregnant women and the first to be used in the Cape Coloured community in South Africa. Given the high prevalence of inadequate choline intake in this and other communities worldwide and the growing evidence that maternal choline supplementation may benefit infant neurodevelopment and can mitigate adverse effects of prenatal alcohol exposure, this tool may be useful for both research and for future clinical outreach programs aimed at identifying pregnant women at risk for choline deficiency. The Choline-QFFQ development and validation methodology we employed can be used in other communities as well, which may be of significant public health utility given the widespread public health burdens of dietary choline inadequacy and maternal alcohol consumption during pregnancy. Our findings demonstrating validity of the QFFQ for use in this highly disadvantaged, poorly educated population supports its potential utility for use in a broad range of social contexts.