Non-citizens often face barriers to HIV care and treatment. Quantifying knowledge of positive HIV status and antiretroviral therapy (ART) coverage among non-citizens in a high HIV-prevalence country like Botswana that is close to achieving UNAIDS “90-90-90” targets may expose important gaps in achieving universal HIV testing and treatment.The Botswana Combination Prevention Project (BCPP) is a pair-matched cluster-randomized trial evaluating the impact of prevention interventions on HIV incidence in 30 rural or peri-urban communities. Community case finding and HIV testing were conducted in home and mobile venues in 15 intervention communities from October 2013-September 2017. In this secondary analysis, we compared HIV positivity, knowledge of positive HIV-status, and ART status among all citizens and non-citizens assessed at intake in the intervention communities.HIV status was assessed in 57,556 residents in the intervention communities; 4%  were non-citizens. Five communities accounted for 81% of the total non-citizens assessed. A lower proportion of non-citizens were HIV-positive  compared to citizens  [p = 0.026]; however, a larger proportion of non-citizens did not know their HIV-positive status prior to BCPP testing (75%) as compared to citizens (15%) [p = 0.003]. Among residents with knowledge of their HIV-positive status before BCPP, 79% of the non-citizens (72/91) were on ART compared to 86%  of citizens (p = 0.137).Although non-citizens were less likely to know their HIV-positive status compared to citizens, there were no differences in treatment uptake among non-citizens and citizens who knew their status. Designing interventions for non-citizens that provide HIV testing and treatment services commensurate to that of citizens as well as targeting communities with the largest number of non-citizens may help close a meaningful gap in the HIV care cascade and ensure ethical treatment for all HIV-positive persons.NCT01965470 (Botswana Combination Prevention Project).ClinicalTrials.gov:  Knowing one’s positive HIV status and initiating and staying on treatment are essential for combatting the HIV epidemic. The UNAIDS “90-90-90” targets establish goals for 90% of all HIV-infected persons to know their status, 90% of persons with diagnosed HIV infection to receive sustained antiretroviral therapy (ART), and 90% of all persons receiving ART to be virally suppressed by 2020 . AchieviBotswana, a high HIV prevalence country with 22.8% of its adult population infected with the HIV virus , is currOne subpopulation in Botswana who may not be adequately covered by the HIV care cascade is the non-citizen population that is currently residing in the country. Migratory sub-populations may experience poor health outcomes and contribute to the ongoing epidemic because of reduced access to HIV testing and treatment programs designed to serve their unique needs . BotswanThe largest cities in Botswana attracting migrant labor are geographically close to the aforementioned neighboring countries with high HIV prevalence. Migrant workers becoming infected upon reaching Botswana may be less willing to access HIV testing given that they are not eligible for free treatment. In addition, HIV-positive non-citizens receiving treatment in their country of origin may be unlikely to return for prescription refills and ongoing health care and, thus, are at risk for developing drug resistance. Consequently, Botswana may have a substantial group of people not receiving WHO and UNAIDS recommended universal HIV testing and treatment to reduce mortality among infected persons and to promote epidemic control.There are limited data on the extent of HIV infection within the non-citizen population in Botswana , and theThe Botswana Combination Prevention Project (BCPP) is a cluster-randomized HIV prevention trial evaluating the impact of a package of prevention interventions on population-level HIV incidence in 30 rural or peri-urban communities in Botswana with an average population of 6,000 per community. Fifteen communities were randomly assigned to the intervention arm and 15 to the arm receiving standard of care services. A full description of the study design is available elsewhere . HIV andAll community residents 16–64 years old were interviewed and assessed for HIV status after providing verbal consent as per Botswana’s HIV testing guidelines for anyone aged ≥16 years. . CommuniCommunity campaigns composed of both home and mobile testing strategies were employed to reach all community residents aged 16–64 years. For the home-based strategy, counselors received lists of all household plots for each of the intervention communities (obtained from Google maps and Botswana 2011 census data). GPS coordinates were used to locate the households on handheld tablets. Counselors returned to households up to three times in order to find residents at home for assessment of HIV status and HIV testing if indicated. The study teams conducted evening and weekend HIV testing and made appointments for home-based testing for times when absent residents would be home. Mobile testing was made available throughout communities at high traffic locations including markets and transport hubs. In addition, mobile testing was provided at community events, health fairs, work sites and shebeens/bars. Persons accessing mobile venues completed the same study procedures as persons participating in the home. All HIV infected persons not on ART identified in home or mobile venues were counseled on the benefits and importance of early care and treatment for their health and the health of partners and children.All responses to structured interview questions and results of HIV tests were collected on encrypted handheld tablets, which were synchronized daily with the research database and removed from the tablet after synchronization to maximize data confidentiality.For field-based rapid HIV testing, finger stick testing using Unigold  and KHB  tests was conducted following the Botswana national testing algorithm. Quality control was assured by re-testing samples from each community in the reference laboratory using enzyme-linked immunosorbent assay (ELISA) and HIV-1 Western Blot testing.Algorithms were designed to measure three main outcomes: HIV positivity, knowledge of positive HIV status prior to BCPP, and ART coverage.HIV-positive status was defined as a positive result on an HIV test conducted through BCPP or documentation of HIV positivity at intake  of unique individuals found in either home or mobile venues.Knowledge of positive HIV status prior to BCPP intake was defined as the show of documentation of HIV positivity prior to BCPP interview date .Current on ART was defined by documentation of current use of ART  at the time of BCPP intake.SAS 9.4 was used for all statistical analyses. SAS PROC SURVEYFREQ was used to produce frequencies adjusting for clustering of observations within communities. Bivariate tests of association were conducted using Rao-Scott Chi-square tests, with a critical alpha level of 0.05. SAS PROC SURVEYLOGISTIC was used to test for association between sociodemographic factors of interest and HIV positivity, prior knowledge of HIV-positive status and ART status. These models produced unadjusted and adjusted odds ratios (ORs) and 95% confidence limits.The study was approved by the Centers for Disease Control and Prevention Institutional Review Board (Protocol #6475) and the Botswana Health Research and Development Committee . The study was monitored by an Independent Data and Safety Monitoring Board.BCPP interviewed 60,139 residents in the intervention communities. Among those interviewed, HIV status was ascertained in 96%  of residents  with only 4% refusing to be tested or show documentation of their HIV status. Overall, 4% (n = 2463) of the assessed residents self-reported as non-citizens. The refusal rate among non-citizens was higher than the refusal rate among citizens .Among all residents assessed, 21%  were HIV-positive. Non-citizens represented 3% (n = 369) of all HIV-positives identified. Five of the 15 communities  accounted for 83% of the HIV-positive non-citizens identified. Four out of five of communities were located just outside of Francistown, the second largest city in Botswana, which is close to the border of Zimbabwe. The proportion of residents who were HIV-positive ranged from 9% to 28% across communities among non-citizens and 14% to 29% among citizens.A smaller proportion of non-citizens were HIV-positive  as compared to citizens  [p = 0.026]. Of the 11,785 HIV positive persons identified, 83%  had documentation of being HIV-positive and 17%  tested HIV-positive in BCPP. A significantly smaller proportion of non-citizens knew their HIV-positive status prior to BCPP testing (25%) than did citizens . Among all HIV-positives, 71%  were currently on ART. Among HIV-positive non-citizens who knew their status prior to BCPP, 79% (72/91) were on ART compared to 86%  of citizens with prior knowledge of their HIV status (p = 0.137). The results presented in this paper offer insight into the HIV epidemic among non-citizen residents of Botswana and the gaps in coverage of vital HIV services for individual health and epidemic control. Among residents assessed, HIV positivity among non-citizens was 15%. Only 25% of HIV-positive non-citizens knew their HIV status and thus the vast majority of non-citizens were not receiving treatment. Among non-citizens who knew their positive status, 79% were receiving ART, a percentage similar to ART use among citizens (86%). In sum, these findings suggest that this important sub-population in Botswana has substantial need for HIV services but is critically underserved.Data from extensive community-based HIV case finding and testing services showed that non-citizens accounted for 4% of the overall population in the 15 intervention communities. However, five communities accounted for 81% of the non-citizens assessed and 83% of the positive non-citizens identified. These findings suggest that HIV interventions for this sub-population should be targeted to specific geographic locations which are most likely to attract migrant labor and/or refugees.Botswana offers free HIV testing to non-citizens, but at BCPP intake only 25% of non-citizens knew they were HIV-positive compared to 85% of citizens. Non-citizens may not proactively seek HIV testing for a myriad of reasons related to their immigration status including disrupted living situations, lack of support from family members, negative attitudes of healthcare workers, fear of deportation, stigma, and lack of availability of HIV treatment even if they know their status. Evidence in other studies have shown similar factors to be challenges for immigrants accessing healthcare in Southern Africa ,14.Despite the low level of knowledge of HIV status at intake, 94% percent of non-citizens offered an HIV test consented to testing, and many sought testing services in mobile venues. More non-citizens were assessed in mobile venues than in the home, but more HIV-positive non-citizens were identified in the home. These findings suggest that non-citizens are receptive to HIV testing, and both home and mobile testing services reach a high proportion of non-citizens so should be part of the testing strategy for reaching this group.Unlike the HIV-positive citizens, the majority of HIV-positive non-citizens were not on ART when first encountered by BCPP. Treatment options are likely cost-prohibitive for many within this group and they are not eligible for government-subsidized ART. Thus, the health of HIV-positive non-citizens is probably greatly compromised and viral suppression coverage in the community as a whole is unattainable. If non-citizens access ART in their countries of origin and experience treatment interruptions due to cost when they migrate, this may introduce drug mutations with implications if the resistant virus is further transmitted.In the Southern African Development Community (SADC) that includes Botswana, there are concerns that the HIV response in the region does not adequately address the movement of healthcare users within and between countries thereby impacting universal test and treat programs. In a reSeveral methodological limitations existed within the study. Measuring the non-citizens’ knowledge of positive HIV status prior to the BCPP intervention was limited to the individual’s ability to produce documentation of a prior HIV test result. Without the government-issued Omang, the study team was unable to obtain any clinical data on HIV testing and ART status for non-citizens using a unique identifier as was possible with the citizen group. Clinical records  were used to augment the documentation of intake data among the citizens but unavailable for the non-citizens. Therefore, it is possible that a higher proportion of non-citizens were “known HIV-positive” at intake than reported. Another methodological limitation to the study was that BCPP was conducted in rural and peri-urban communities and not the larger cities which serve as economic hubs and typically draw immigrants seeking work in Botswana. Therefore, rural/peri-urban communities may not be representative of where most non-citizens reside in Botswana. This design feature could explain the difference between the estimated 7% non-citizen population in Botswana and BCPP’s report of 4% non-citizenship among all community residents assessed for HIV. Thus, the HIV positivity and treatment rates found among non-citizens may or may not be generalizable to the country as a whole.Non-citizens have lower levels of knowledge of positive HIV status compared to citizens; thus, overall ART coverage is low in this sub-population in Botswana. When they know their HIV-positive status, rates of ART uptake are similar among citizens and non-citizens. In a country close to achieving their “90-90-90” goals but still reporting new HIV infections, it is imperative to examine gaps in the HIV care cascade at a sub-population level. Increasing access to HIV care by eliminating social and monetary barriers for non-citizens may improve health outcomes for all and possibly contribute to epidemic control in Botswana. Focusing intervention efforts on the geographic locations where most non-citizens reside may produce the greatest benefit.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(CSV)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Heparin and heparan sulfate (HS) are polydisperse mixtures of polysaccharide chains between 5 and 50 kDa. Sulfate modifications to discreet regions along the chains form protein binding sites involved in cell signaling cascades and other important cellular physiological and pathophysiological functions. Specific protein affinities of the chains vary among different tissues and are determined by the arrangements of sulfated residues in discreet regions along the chains which in turn appear to be determined by the expression levels of particular enzymes in the biosynthetic pathway. Although not all the rules governing synthesis and modification are known, analytical procedures have been developed to determine composition, and all of the biosynthetic enzymes have been identified and cloned. Thus, through cell engineering, it is now possible to direct cellular synthesis of heparin and HS to particular compositions and therefore particular functional characteristics. For example, directing heparin producing cells to reduce the level of a particular type of polysaccharide modification may reduce the risk of heparin induced thrombocytopenia (HIT) without reducing the potency of anticoagulation. Similarly, HS has been linked to several biological areas including wound healing, cancer and lipid metabolism among others. Presumably, these roles involve specific HS compositions that could be produced by engineering cells. Providing HS reagents with a range of identified compositions should help accelerate this research and lead to new clinical applications for specific HS compositions. Here I review progress in engineering CHO cells to produce heparin and HS with compositions directed to improved properties and advancing medical research. Heparin and heparan sulfate (HS) are related polysaccharide chains with expression and structural similarities that give them similar properties but with some fundamental differences. Heparin and HS polysaccharide chains have backbones composed of the same repeating disaccharides that are, in both cases, modified by sulfation at various positions of the sugar residues. Biosynthesis of heparin and HS—initiation, elongation, and modification of the polysaccharide chains—proceed through the same enzymatic pathway in the Golgi , 2. WherIn vivo heparin function is much less well-understood. Heparin may aid in the packaging and storage of histamine and other inflammatory mediators stored in mast cell granules that are released upon IgE stimulated degranulation. Structurally, heparin can be thought of as over-sulfated HS as it tends to be considerably more highly sulfated; including the high frequency of a characteristic trisulfated disaccharide, and the presence of the antithrombin 3 (AT3) binding site (see below) that is almost completely absent in HS. This structure is responsible for the potent anticoagulant properties for which pharmaceutical heparin is known and widely prescribed. Pharmaceutical heparin is a highly purified fraction of material prepared from animal tissue, largely porcine intestine, and formulated for intravenous or subcutaneous administration. in vivo heparin is sequestered in granules where it is not thought to have effects on blood clotting, although, on release, there may be protective effects on tissues from inflammatory cell invasion  are gaining acceptance and market share because of more convenient routes of administration, decreased monitoring requirements and in some cases better safety profiles. How extensive this market infiltration becomes remains to be seen. Heparin is fast acting, completely reversible and currently has the advantage of efficacy and safety data from dosing patients globally for over 75 years. There are also clinical situations where heparin may be preferable, such as for patients with prosthetic heart valves as NOA have greater risks of valve thrombosis , 15, prePerhaps the real power of cellular production is the prospect of altering the structure of heparin, in order to improve its properties, or to produce polysaccharide chains that are tailored to the non-anticoagulant applications identified retrospectively. For example, in cardiopulmonary bypass surgery where heparin is the drug of choice , risks oBiotechnology has come a long way in eliminating the need to source medicines from animals. Heparin has been an exception, perhaps due to the large number of slaughtered pigs in China and the low (but rising) cost of labor there, but more importantly, producing recombinant heparin entails a higher level of complexity than producing a recombinant protein. Unlike recombinant proteins that are expressed by a single gene, heparin is synthesized in a complex metabolic pathway involving over 20 enzymes. Heparin polysaccharides are uniquely produced in mast cells but because mast cells are particularly difficult to propagate and maintain, they are not suitable for commercial production. The CHO cell line is an industry standard for producing recombinant therapeutic products. This familiarity may be an advantage from a regulatory standpoint, but another advantage is that CHO cells make relatively large amounts of HS. Producing recombinant heparin from CHO cells entails engineering the CHO cells genetically, to produce HS with the anticoagulant properties of heparin. Further genetic engineering could be aimed at reducing other protein binding substructures, for example, platelet factor 4 (PF-4) binding to reduce the risk of heparin-induced thrombocytopenia (HIT).Two major challenges confront the prospect of developing cell lines that produce recombinant heparin. The first challenge is the difference in composition between heparin and HS. Heparin inhibits coagulation by binding to AT3 and enhancing its activity ~1,000-fold). AT3 is a serine protease inhibitor that inhibits blood clotting by neutralizing thrombin (Factor IIa), Factor Xa and other serine proteases in the coagulation pathway. Heparin binds to AT3 via pentasaccharide binding motifs located along the heparin chains  residues by two enzymes, GlcNAc and GlcA transferases , to the N-position of N-acetylglucosamine (four isozymes NDST1-4), to the 3-O-position of N-sulfoglucosamine (seven Hs3st isozymes), and to the 6-O-position of N-acetyl/sulfoglucosamine (three isozymes Hs6st1-3) (Heparin and HS are members of a class of polysaccharides referred to as glycosaminoglycans (GAGs). The heparin/HS backbone is a polysaccharide synthesized as a copolymer of alternating D-glucuronic acid (GlcA) and s6st1-3) , 23.First, NDST deacetylates and sulfates a subset of GlcNAc residues to form GlcNS.N-deacetylation and sulfation of GlcNAc is preferred in regions proximal to GlcNS.Further The epimerase requires an upstream (toward the non-reducing end) GlcNS.O-sulfation.The epimerase is inhibited by downstream (toward the reducing end) 6-O-sulfation generally requires prior N-sulfation or an adjacent GlcNS unit.6-O-sulfation occurs in areas of high sulfate and iduronic acid.3-N- and 6-O-sulfation in the absence of 2-O-sulfation.Sulfation compensation tends to maintain the wildtype net charge e.g., by increasing Sulfation levels are determined by the expression levels of the modification enzymes but how that is regulated is not well-known. Synthesis is not template driven, and none of the reactions in the biosynthetic pathway proceed to completion so there is structural heterogeneity among the polysaccharide chains in both size and structure. Typically, heparin and HS samples are characterized by compositional analyses, which quantify the disaccharides that make up the polysaccharide chains by their modifications. Table O-sulfated group on the central glucosamine (residue 3) and a critical 6-O-sulfate group on the non-reducing end glucosamine (residue 1). These two sulfate groups are responsible for more than half of the binding energy of the interaction  suggests that increasing 6-O-sulfation by over expression of Hs6st1, Hs6st2, Hs6st3, or combinations may be required to achieve the USP standards for heparin. This study also shows that it will be important to achieve a balance of expression levels between transgenes and endogenous genes. In E. coli and Saccharomyces cerevisiae, the expression levels of enzymes involved in metabolic pathways have been controlled by gene titration, promoter engineering, or transcriptional regulation  . One appnditions –42 relatnditions . Heparinnditions , 44 althnditions . To overnditions . Bioreacnditions –50. Altenditions . Lyase dnditions , 53. Thinditions .The second major challenge to produce a commercially viable product is achieving production levels that can compete economically with the current production of animal derived heparin. Hundreds of millions of slaughtered pigs in China translates into low priced heparin. Industry production methods are closely guarded secrets so determining the cost of heparin production is difficult. Online prices for heparin through Pharmacy Checker.com and GoodRx show retail prices ranging from $0.023–$5.50/international unit (IU) for quantities between 10,000 and 60,000 IU. Costs for recombinant heparin API production come primarily from initial equipment outlay operation and maintenance, cell culture media, and supplies and purification and processing. Increasing production efficiency (or decreasing costs) will be critical. Not considering overhead and sales & marketing, we estimate that recombinant heparin production becomes competitive at production levels of 1 g/L.As HS is produced as a proteoglycan, one way to increase production may be to overexpress the core protein. Mast cells produce heparin on the core protein serglycin, which is not normally made in CHO cells , 56. In As discussed, non-anticoagulant physiological properties of heparin and HS have been identified retrospectively from patients treated with heparin to prevent blood clotting . These pRelating HS structure to function is complex because carbohydrate chemistry makes sequencing difficult and HS is a heterogenous mixture of polysaccharide chains. Currently HS is characterized by its composition of sulfated disaccharides despite the fact that the binding sites are families of higher order oligosaccharides that are not revealed by the compositional analysis. HS function is determined by the protein binding sites in localized regions along the chains, but the long polysaccharide chains may contain multiple protein binding sites some of which may stimulate while others inhibit particular cell signaling pathways. This complexity is compounded by the heterogeneity, presumably enabling HS from a particular tissue to be involved in multiple cell signaling pathways. None-the-less HS participates in defined physiologically relevant processes which appear to be regulated by the expression and activity of the enzymes in the metabolic pathways. From an applications standpoint, using HS therapeutically may depend on producing HS with a composition tailored to the relevant HS binding proteins or put another way with the right combination of protein binding sites. That composition reflects a particular combination of binding sites may be reflected by the fact that tissue specific compositions are conserved among different species.O-sulfation. At the cell surface, HS typically functions as a coreceptor modifying growth factor affinity/activity albeit more subtly than the growth factor receptor peptides; much like a rheostat (HS acts by binding proteins in the extracellular matrix (ECM) and growth factors and cytokines at the cell surface. In the ECM, it may provide a growth factor sink, which could be released by digestion with heparanase or the activities of other heparin/HS modification enzymes such as the SULFs which reduce subsets of 6-rheostat . Bindingrheostat . In somerheostat , 61. Othrheostat . As varirheostat , 63. By rheostat .Through research and clinical observations, a number of potential non-anticoagulant clinical applications of heparin/HS have come to light. In acute inflammatory reactions, heparin has been shown to protect against ischemia-reperfusion injury in animal models , 66. HepThere is also significant evidence for the benefit of heparins in cancer because of the frequent use of heparin in the prophylaxis of venous thromboembolism in cancer patients , 79–85. In these applications, anticoagulation is usually an unwanted side effect. In addition, heparin is highly sulfated and is therefore likely to influence multiple signaling pathways, some of which may be counterproductive. Thus, engineering HS may provide advantages due to the lack of anticoagulant activity and because the composition can potentially be engineered to tailor it to the application. It is thus important to understand the relationship between HS composition and application. Examples discussed below are from research aimed at determining how HS composition affects physiology and pathophysiology, which could lead to therapeutic HS compositions or applications where HS is a drug target.N-sulfation, 6-O-sulfation and 2-O-sulfation was associated with the macrophages in a perpetual activated state, with increased proinflammatory cytokine secretion, enhanced infiltration and foam cell conversion leading to atherosclerotic lesion formation and increased diet-induced obesity. Macrophages constitutively express low amounts of Type I IFNs. Perpetual macrophage activation in the knockout suggests that the capacity of HS to bind Type I IFNs is a mechanism to retain the interferons near their site of secretion preventing them from interacting with their receptors IFNAR1 and IFNAR2. In this model, under resting conditions, highly sulfated macrophage HS maintains Type I IFN in a quiescent state through sequestration of IFN-β. This model is supported by observations that HS sulfation is decreased in advanced atherosclerotic lesions  are important regulators of macrophage stimulation by Type 1 interferon (IFN) . In the  lesions , 95 and  lesions . A simil lesions , 98. WntO-sulfation  because DNA and heparin compete for binding sites on NE (O-desulfated heparin chains should be in the range of 15 saccharides.In cystic fibrosis (CF), high levels of neutrophil elastase (NE) in lung and sputum promote inflammation by up-regulating neutrophilic cytokines, help disrupt the protease-antiprotease balance by activating other proteases and degrading antiproteases as well as elicit a series of additional pathological effects that make NE an established biomarker of CF progression –105. Seres on NE . PolymerO-sulfation is critical for inhibition of the selectin interactions   . Reductig assays . Althougg assays . In lighA separate study identified a hexasaccharide that selectively inhibits cancer-stem cells (CSCs) . Like thN-sulfation also lack 2-O-sulfation as 2-O-sulfation depends on N-sulfation (see above) however the cells maintain half of the normal 6-O-sulfation. These cells maintain their ESC characteristics under conditions that maintain pluripotency however angiogenic differentiation is impaired. Thus, sulfated HS is required for normal development. Knocking out 2-O-sulfation resulted in an increase in N-and 6-O-sulfation with proliferative defects and defects under conditions promoting neural differentiation. Under these conditions haemopoietic differentiation appeared normal. Consistent with the increase in 6-O-sulfation, neural defects were also observed in SULF enzyme mutants, that also increased 6-O-sulfation. However, under these conditions, defects in haemopoietic differentiation were also evident. This may suggest the need for tighter control of 6-O-sulfation  have been used to characterize the role of HS in regulating differentiation because alterations in HS structure can be lethal in mice . ESCs arulfation .O- and N-sulfation were identified with HS being more effective than heparin; again indicating that the organization of the sulfate groups on the polysaccharide chains is important, not just high levels of sulfation. Neural differentiation required some distinctly different structures. Addition of heparin/HS to HS-containing cells significantly improved neural differentiation where N-, 2-O- and 6-O-sulfations were all important. Thus, there is a strict requirement to specifically tailor the composition of exogenous HS added, depending on the type of differentiation  in the aging mice exhausted the quiescent SCs leading to fibrosis and sarcopenia (Satellite cells (SC) are muscle stem cells that lie quiescent in muscle fibers. In response to injury, SCs are activated, proliferate as myoblasts and form new muscle fibers . Understrcopenia .N-sulfated; 2-O-sulfated; 6-O-sulfated; N- and 2-O-sulfated; N- and 6-O-sulfated; N-, 2-O- and 6-O-sulfated and over-sulfated heparin with additional 3-O-sulfation. All of the mimetics except over-sulfated heparin reduced differentiation. In most cases, decreased differentiation was accompanied by increased proliferation. Over sulfated heparin did not reduce differentiation but reduced cell numbers. One mimetic: N-acetylated-2-O-desulfated (with increased 6-O-sulfation like aging) inhibited differentiation and did not promote proliferation. Since myoblast proliferation is strongly promoted by FGF2, over-sulfated heparin was tested to see if its antiproliferative effects were mediated by inhibition of FGF2 signaling. Over-sulfated heparin reduced Erk1/2 phosphorylation presumably by acting as an extracellular sink for FGF2. In contrast, N-acetylated heparin which inhibits differentiation, prolonged Erk1/2 phosphorylation consistent with promoting cell expansion. These results strongly suggest that myoblast proliferation is regulated by HS though FGF2 signaling. The HS mimetics were then tested for their ability to promote or inhibit SC self-renewal since aging is accompanied by reductions in SCs. In cell culture, self-renewal consists of myoblasts that exit the cell cycle but instead of differentiating they enter a quiescent state. Quiescence can be detected by the expression of transcription factor Pax7  the GAGome library of engineered CHO cells was used to display complex GAG features and determine which features are relevant for specific binding interactions and the biosynthetic enzymes responsible for their production . SimilarA number of strategies for producing recombinant heparin have been reported. In an attempt to increase production, HEK293 and HEK293T cells were transfected with human serglycin; His-tagged to facilitate analyses of the attached GAG chains . Eighty-E. coli K5  12-mer-1 displays anticoagulant activity similar to LMWHs, is protamine-reversible and is amenable to multigram-scale chemoenzymatic synthesis . Scale uTEGA's strategy is to engineer CHO cells to produce recombinant heparin/HS, which enables the entire supply chain to be under GMP control. With cell sourced HS, we achieve a high level of structural consistency in size and composition of the polysaccharide chains. Like the other strategies for producing recombinant heparin/HS, achieving sufficiently high and cost-effective production levels will be a challenge. Some of this may involve bioprocess development and scale-up along with cell engineering. We hope to accelerate the cell engineering process by introduction of multiple genes simultaneously and then using high throughput screening and analysis methods we have developed.The author confirms being the sole contributor of this work and has approved it for publication.in vitro Heparin and Heparan Sulfate Compositions and Methods of Making and Using 12/15/17 TEGA Therapeutics, Inc.CG was employed by company TEGA Therapeutics, Inc. Work reported in this article is covered under patent applications: 1. Patent applications: PCT/US2016/067373 Cellular Glycosaminoglycan Compositions and Methods of Making and Using 12/16/2016 TEGA Therapeutics, Inc. 2. PCT/US2017/066860


The 19th Annual Advances in Genome Biology and Technology (AGBT) meeting came back to Marco Island, Florida, and was held in the renovated venue from 27 February to 2 March 2019. The meeting showed a variety of new technology, both in wet lab and in bioinformatics. This year’s themes included single-cell technology and applications, spatially resolved gene expression measurements, new sequencing platforms, genome assembly and variation, and long and linked reads. TREM2, which mediates DAM activation. Interestingly, while normal mice show only one type of microglia, humans show seven, which most likely represent different activation states. Jiannis Ragoussis of McGill University  described a combination of single-cell copy number variation and single-cell 3′ end expression analysis. Using approximately 80,000 single cells from glioblastoma and approximately 20,000 single cells from normal fetal brain for single-cell RNA sequencing (scRNA-seq) and 30,000 glioblastoma cells for single-cell DNA sequencing (scDNA-seq), he described a progenitor population including most cycling cells of the cancer. This work suggests the possibility to define therapeutic targets for glioblastoma stem cells. Manolis Kellis from MIT  described single-cell analyses of postmortem brains in Alzheimer’s disease. He found early cell-type-specific alterations but late global changes and large sex differences that increased with disease progression. He showed that both somatic mutations and cell type proportion changes underlie disease phenotypes, and that distinct epigenomic and transcriptional signatures underlie different sub-phenotypes of Alzheimer’s disease, pointing to much underappreciated complexity. Georg Seelig, of the University of Washington , presented his laboratory’s work on split-pool ligation-based transcriptome sequencing (SPLiT-seq). This technology uses several rounds of combinatorial barcoding to profile a large number of fixed cells or nuclei  without the need to isolate each cell, by (i) making cDNA within each cell and (ii) repeatedly barcoding the cDNAs of a few (approximately 1000) randomly chosen cells with the same barcode and repooling the cells. Application of SPLiT-seq to mouse brains at postnatal days 2 and 11 revealed more than 100 cell types. Cole Trapnell, also of the University of Washington, talked about single-cell expression profiling of whole embryos, measuring more than one million single cells simultaneously to describe mammalian organogenesis, using novel tools such as Monocle3, which is still under active development. This revealed cell populations and trajectories that appear difficult to delineate with smaller cell numbers. He furthermore described approaches to measure the impact of single-cell perturbations on hundreds of thousands of single cells and their trajectory, a technology termed “Sci-Chem”. Hagen Tilgner, one of the authors of this report, showed non-random pairing of distant exons in full-length isoforms and a method to monitor complete isoforms in thousands of single cells . This revealed isoform expression across neuronal and glial subtypes. Jason Underwood of Pacific Biosciences  presented a similar method, called single-cell ISO-Seq (ScISO-Seq), and its application to human and chimp brain organoids. He revealed polyadenylation signals that are consistent with the literature and unknown introns within 3′ UTRs that would be expected to trigger nonsense-mediated decay. Both ScISOr-Seq and ScISO-Seq use microfluidics for cDNA barcoding and long reads to deduce isoforms in single cells.Aviv Regev of the Broad Institute  classified progress in the single-cell world into measurements, inference, and perturbations. She showed (among other topics) her lab’s work in the cellular architecture of ulcerative colitis, searching for “cells of action” rather than simple genome-wide association study (GWAS) hits. Leveraging sequencing of approximately 120,000 colon cells, her lab revealed about 50 types of cells and a new cell type: the inflammatory fibroblast. Interestingly, the expression of most ulcerative colitis risk genes obtained from GWASs is also specific to particular cell types. Hadas Keren-Shaul of the Weizmann Institute  discussed the discovery of disease-associated microglia (DAM). In a single-cell comparison of immune cells of wild-type mice and an Alzheimer model, she found three classes of microglia, two of which do not occur in wild-type animals. These DAM overexpress a number of genes, including As in AGBT 2018, spatial profiling of molecules continued to play a prominent role. In particular, Katherine McNamara of Stanford University  reported on her use of the NanoString GeoMx™ technology to profile RNA and proteins in Her2-positive breast cancer samples. She found tumor heterogeneity to increase and HER2 signaling to decrease with targeted therapy. The Broad Institute’s Sanja Vickovic reported on spatially resolved simultaneous measurements of polyadenylated host RNA and 16S bacterial RNA. She measured 11,000 host (mouse) genes and identified nine types of bacteria. In addition, she also presented a new method for in situ RNA barcoding at the subcellular 2-μm level termed high-density spatial transcriptomics (HDST). Evan Macosko, also of the Broad Institute, described the novel method of Slide-seq. Slide-seq generates cDNAs that are barcoded for their position on a slide of frozen tissue and currently achieves a resolution of 10 μm. The Macosko and Chen labs used Slide-seq to investigate mouse hippocampus and cerebellum, describing, for example, spatially restricted gene expression patterns in the Purkinje layer of the cerebellum. This spatial division was also apparent for other cerebellar cell types, including Bergmann glia and granule cells.PacBio reported the new Sequel II system and on advances of circular consensus reads (CCS), which shows qualities rivaling those of Illumina. For genome assembly of the HG002 sample (Genome In A Bottle), this yields N50 s of 16.0–18.0 Mb and eases copy-number variation detection. Genapsys  presented a soon to be released short-read sequencing platform. This product is low throughput and low cost  but achieves high substitution and indel accuracy, assumingly targeting the precision health market. Its low cost could potentially also be valuable in smaller laboratories for targeted variation analysis. On the other end of the spectrum , MGI Americas  showcased the MGISEQ-T7 sequencer, a technology “created” for a world in which every individual on earth would be sequenced in the next 50 years.Mike Schatz of John Hopkins University  discussed the importance of the role of structural variants (SVs) in tomato genomes in phenotypic differences. For tomatoes, a reference genome became available in 2012, but few large-scale studies followed. Schatz and colleagues identified 100 samples and sequenced them using PacBio and Nanopore long-read sequencing to study the landscape of SVs. Analysis of the first 80 long-read sequenced genomes reports about 15,000–50,000 SVs dominated by insertions and deletions. Other complex SV types are also present in their data. Arend Sidow of Stanford University described a reference-assisted assembler to detect SVs using linked reads. Short-read methods miss a large portion of genome-wide SVs. His method characterizes complex SVs missed by short reads and works well for small insertions sized less than 500 base pairs. Sidow ended his presentation with a sublime analogy: a $20,000 sedan, a $40,000 SUV, and a super luxury $400,000 car, resembling the relative cost and utility of standard Illumina short fragments, 10× genomics long fragments, and PacBio CCS, respectively, to conclude that the 40,000 SUV can get the job done.Drosophila studies, Andy Clark of Cornell University  reported on repetitive elements and their variation among fly species. While we have come to think of repetitive sequence as long-read territory, he explained how all sequencing technologies, including Illumina, contain repeat information, which can be extracted by counting k-mers. This revealed that some repeats are equally abundant among fly species, but for others a 100-fold difference can be observed, with evidence for selective constraints on gene expression. Richard Green of University of California, Santa Cruz  showed his lab’s work on detecting archaic human ancestry algorithmically. They find that for approximately 10% of the modern human genome, one doesn’t find any trace of archaic genome influence. Approximately 1.5% of these human-specific regions have human-specific derived genetic variants. This represents regions that contribute to the differences between Homo sapiens and Neanderthals/Denisovans. One such region contains the splicing factor NOVA1, in which a Neanderthal valine amino acid is replaced by a Homo sapiens isoleucine. Organoid work suggests that this causes a splicing difference in protocadherin and other genes. Beryl Cummings of the Broad Institute leveraged the vast amount of genotype-tissue expression (GTEx) RNA-seq data in order to derive transcript-level isoform quantifications for the interpretation of putative loss-of-function variation. She finds that genetic variation in highly expressed exons is biased towards low-frequency alleles. Leveraging this expression data allowed her to filter wrongly annotated loss-of-function variation, with little impact on trustworthy pathogenic variation. Tuuli Lappalainen of the New York Genome Center  reported on the use of allele-specific expression, emphasizing that allele-specific data are less affected by confounders than other approaches. She showed how the ANEVA Dosage Outlier Test  captures large rare-variant effects on transcription and can be used to prioritize genes. She captures various different kinds of variants, including those introducing stop codons or frame-shifts as well as those altering splicing patterns.Reporting on Mikhail Kolmogorov, a PhD student at University of California, San Diego , described Flyer, a new assembly method for error-prone long reads. He argued that the main issue in genome assembly is the characterization of all repeat families. Flyer is based on a novel repeat graph reconstruction, and similar to most long-read assemblers does not utilize a k-mer-based de Bruijn graph. He presented an algorithmic approach which results in a more accurate repeat graph. Compared with competing tools , this novel approach significantly reduces the number of misassemblies. Adam Phillippy of the National Human Genome Research Institute  described work on a gapless human X chromosome assembly as the first human genome assembly contains hundreds of gaps in unresolved regions. To resolve this he used the, essentially haploid, CHM13 human cell line and long/linked reads, including 40× coverage with ultra-long Nanopore reads. Some reads exceeded 1 Mb and approximately 44 Gb of sequences were reads of over 100 kb. Combining their high-coverage Nanopore data with 70× coverage PacBio data, together with additional manual steps, he presented a gapless assembly for the human X chromosome and a promising whole-genome assembly (NG50 exceeding GRCh38). Karen Miga of UCSC described her effort in building high-quality reference human genomes using PromethION, the new Nanopore sequencer. They picked ten offspring from a diverse set of offspring–parent trios. Miga mentioned that the projected cost of each individual genome is about $10,000, which is lower than the cost of previous generations of long-read techniques but still one order of magnitude higher than standard short-read sequencing. She presented a pipeline for processing individual genomes from sample extraction to assembly that can be completed in one week, with the aim to produce near-gapless reference whole genomes. Edward Rice of University of Nebraska  spoke about chromosome-length haplotigs for a hybrid cattle/yak assembly. He discussed the importance of finishing genomes and showed that 378 gaps remain in a recently improved cattle genome. His main focus was the assembly of diploid genomes using trio-binning to separate complete haplotypes. Trio-binning uses short reads from parental genomes to cluster offspring long reads into the right maternal or paternal haplotype. Rice showed that high heterozygosity eases trio-binning and presented an accurate diploid assembly of an Fl yak/cattle hybrid.CACNA1 genes, which are linked to a variety of diseases. CACNA1C has over 50 exons and spans over 700 kb. Using the MinION to investigate CACNA1C in multiple brain regions, his team reported 28 novel exons and increased total isoform number from about 40 to about 90. Most of the variation in CACNA1C occurs in the intracellular domains. Joanna Kelley of Washington State University  described temporal gene expression profiling of Grizzly bears, which are insulin resistant during hibernation but insulin sensitive at other times. Her team observed a general shutdown of metabolism-related genes during hibernation and now plans to describe isoform abundance during hibernation using PacBio.Wilfried Haerty of the Earlham Institute  reported on complex splicing patterns in the Katherine Pollard of Gladstone Institutes  and University of California, San Francisco  described, among other topics, an approach to understanding protein–DNA binding based on shape rather than sequence. The algorithm is based on Gibbs sampling and finds shape-motifs de novo. She showed how DNA-binding proteins often recognize DNA shape. Interestingly, shape and sequence motifs can be located in proximity, with shape motifs providing specificity. Her lab plans to describe evolutionary conservation of shape motifs in the near future.Based on the presentations at the AGBT this year, large-scale single-cell sequencing is now widely used across different laboratories. Moreover, long-read and linked-read sequencing techniques are applied by different labs for a variety of applications such as isoform detection, de novo assembly, and structural variant characterization. We anticipate hearing about development of additional methods and large-scale studies of long-read and linked-read sequencing at next year’s meeting. Spatial profiling is becoming popular and we expect to see further improvement in technology in the near future.


The cardiovascular (CV) safety in terms of heart failure among different classes of treatment remains largely unknown. We sought to assess the comparative effect of these agents on heart failure outcomes.This study was registered in the International Prospective Register of Systematic Reviews (CRD 42016042063). MEDLINE, EMBASE, and the Cochrane Library Central Register of Controlled Trials were searched. For the primary outcomes reported previously, studies between Jan 1, 1980 and June 30, 2016 were screened, and subsequently updated till Jan 24, 2019. We performed network meta-analysis to obtain estimates for the outcomes of heart failure, in particular by rankograms for ranking of heart failure risk as well as by pairwise comparisons among all classes of anti-diabetic medications.A total of 91 trials were included, among which were 171,253 participants and 4163 reported cases of heart failure events. As for rankograms, the surface under the cumulative ranking curves (SUCRA) of sodium-glucose co-transporters 2 and thiazolidinediones were 93.4% and 4.3%, respectively, signifying the lowest and highest risk of heart failure, respectively. As for pairwise comparisons in the network, sodium-glucose co-transporters 2 were significantly superior to insulin , dipeptidyl peptidase 4 inhibitors , glucagon-like peptide-1 receptor agonists , and thiazolidinediones  in terms of heart failure risk. Furthermore, in an exploratory analysis among subjects with underlying heart failure or at risk of heart failure, the superiority of sodium-glucose co-transporters 2 was still significant.In terms of heart failure risk, sodium-glucose co-transporters 2 were the most favorable option among all classes of anti-diabetic medications.The online version of this article (10.1186/s12933-019-0853-x) contains supplementary material, which is available to authorized users. In 1974, the Framingham studies have reported an approximately two- to five-fold increase for the risk of heart failure in patients with diabetes –5. HowevThis study was registered in the International Prospective Register of Systematic Reviews (CRD 42016042063). The systematic review and meta-analysis was conducted according to the Preferred Reporting Items of Systematic Reviews and Meta-Analyses (PRISMA) guideline . The methttp://www.clinicaltrials.gov were also checked if the registry number was provided. The strategy of searching was provided in Additional file MEDLINE, EMBASE, and the Cochrane Library Central Register of Controlled Trials were searched. For the primary outcomes reported previously, studies between Jan 1, 1980 and June 30, 2016 were screened. We updated the data until Jan 6, 2019 for the outcome of heart failure. Data on To be included, studies had to: be randomized controlled trials; included individuals with type 2 diabetes; compared one kind of anti-diabetic with another or placebo; had at least 1 incident heart failure during follow-up; had a treatment duration of more than 24 weeks. Studies were excluded if they: were crossover studies; non-randomized trials; compared the different dosages or forms of the same drugs; enrolled patients with type 1 diabetes, prediabetes, or insulin resistance.Novel anti-diabetics in our studies referred to the following 3 categories: dipeptidyl peptidase 4 inhibitors (DPP4i), glucagon-like peptide-1 receptor agonists (GLP1a), andsodium-glucose co-transporter 2 inhibitors (SGLT2i). We only included studies on drugs that were already approved by US FDA or European Medicines Agency. Data of the novel anti-diabetic treatment arm were excluded if the dosages were inconsistent with the recommended ones , patients characteristics , intervention details , total number of participants and incidence of heart failure.The methodological quality of included studies was assessed using the tool recommended by Cochrane collaboration . This toNetwork meta-analysis was performed to compare any of the 2 anti-diabetics by incorporating direct and indirect comparison data. The results were reported as odd ratios (ORs) and 95% confidence intervals (CIs). We then generated a ranking of the anti-diabetics in terms of risk of heart failure by calculating the surface under the cumulative ranking curves (SUCRA). Larger SUCRA indicated lower risk of heart failure. Rankograms were also generated .To identify any potential inconsistency between direct and indirect comparisons data, we used a previously described node-splitting method. It separates evidence for a particular comparison into direct and indirect component, excluded one direct comparison at a time, and estimated the indirect treatment effect for the excluded comparison .mvmeta package.All the analyses were performed using STATA version 13 with the Flow chart of study inclusion is shown in Additional file Assessment of methodological quality of included studies is summarized in Additional file Network plot of the comparisons is presented in Fig. Sample sizes and numbers of incident heart failure of included studies are summarized in Additional file Results of the network meta-analysis were summarized in Fig. In our previous work, we have found that novel classes of anti-diabetic agentsshowed favorable CV safety profile in terms of MACE and all-cause mortality, despite small differences between individuals . In the It has been well established that TZDs, when compared with placebo, was associated with an increased risk of heart failure among diabetic patients –31. In oDPP-4 inhibitors and GLP-1 agonists, or collectively termed incretin-based therapies, were found in our network to be respectively taking up 5th and 7th places in rankograms and were both statistically significantly inferior to SGLT-2 inhibitors in pairwise comparisons. For GLP-1 agonists, after LEADER and SUSTAIN-6 trials having demonstrated a reduction in CV mortality but not heart failure hospitalization , 42, subThe complex signaling and mechanisms through which generalized insulin resistance contribute to myocardial dysfunction and ultimately heart failure has recently been reviewed by Riehle et al. . Packer There are several limitations to our current study. First of all, the heterogeneity of trial designs and reporting styles of the included studies, as well as modest event rates in general cannot be overlooked. Therefore, these results were primarily hypothesis-generating in nature and should be interpreted with discretion. Second, information regarding heart failure being that which was with reduced or preserved ejection fraction was virtually indistinguishable in the majority of studies, preventing a deeper layer of exploration in the network which differentiates heart failure clinical phenotypes. Third, since patient-level data was inaccessible to us, other possible variables correlated with heart failure were unable to be accounted for in the current analysis. Ongoing preclinical studies and clinical trials would shed light on how these questions can be answered.Our network meta-analyses demonstrated that, in terms of heart failure risk, SGLT-2 inhibitors were the most favorable option among all classes of anti-diabetic medications; DDP-4 inhibitors and GLP-1 agonists seemed inferior to SGLT-2 inhibitors and metformin. Results from this study indicate that heart failure as a phenotypically distinct entity among diabetic complications may be significantly influenced by choice of glucose-lowering medications, and also call for future studies to further explore the dynamic interplay between pharmacologic effects of different agents, pathognomonic mechanisms of diabetes-related cardiotoxicity, and clinical progression of heart failure.Additional file 1: Table S1. Search strategyAdditional file 2: Table S2. Novel anti-diabetics and recommended dosage.Additional file 3: Figure S1. Flow chart of study inclusion.Additional file 4: Table S3. Baseline characteristics of included studies. Table S4. Methodological quality assessment of included studies. Table S5. Heart failure events of included studies.Additional file 5: Table S6. Potential inconsistency between direct and indirect comparisons assessed by the node-splitting method.Additional file 6: Figure S2. Sensitivity analysis after excluding studies with an arm of fewer than 100 patients.Additional file 7: Figure S3. Sensitivity analysis after excluding studies with a follow-up period of fewer than 48 weeks.


It has been hypothesized that schistosomiasis negatively influences immune reconstitution in people living with HIV starting antiretroviral therapy (ART). In this study, we investigated the effect of schistosomiasis on the course of HIV infection in patients starting ART in a rural part of Tanzania.Retrospective study including patients prospectively enrolled in a HIV cohort in Ifakara, south-central Tanzania between January 1, 2013 and April 1, 2015. Schistosomal circulating anodic antigen (CAA) was assessed in pre-ART cryopreserved plasma. Regression models were utilized to estimate the effect of CAA positivity on virological and immunological failure and a composite outcome of death/loss to follow-up (LFU).At ART-initiation 19.1% (88/461) of patients were CAA-positive. A tendency of higher CD4 increases was seen in CAA-positive patients , 87–285 cells/μl) compared to CAA-negative patients  after 10 months of follow-up. After adjustment for baseline risk factors, CAA-positivity showed no association with virological or immunological failure. In CAA-positive patients, 22.7% (20/88) died or were LFU, compared to 29.5% (110/373) of CAA-negative patients (hazard ratio (HR): 0.76, 95% confidence interval (CI), 0.47–1.22, p = 0.25). After adjustment for age, sex, body mass index, educational attainment, WHO-stage, tuberculosis status, and year of ART initiation, CAA-positivity showed a trend of a decreased hazard of death/LFU , while CD4 count at baseline  and MXD  >1,100 cells/μl  were identified as independently protective factors.Schistosomiasis is prevalent in this HIV cohort and may be beneficial for immunological reconstitution, while no effect on virological failure was apparent. A positive effect of schistosomiasis-induced immunomodulation on survival and retention in care needs confirmation in future studies. Schistosoma) both exert chronic modulatory effects on the host’s immune system. Coinfections, meaning the host is simultaneously infected with both pathogens, are common in sub-Saharan Africa. In this situation the induced immune modulation of one pathogen may affect the course of the disease induced by the other pathogen. One study showed that coinfection with Schistosoma in people living with HIV who begin antiretroviral therapy (ART) may have deleterious effects on the reconstitution of the HIV-induced immunosuppression. In the current study, we investigated the effect of Schistosoma coinfection on the recovery of the patient’s immune system, on the efficacy of ART to suppress HIV replication, and on a combined endpoint of lost to follow-up or death. We found that schistosomiasis may have beneficial effects on immune reconstitution, while no deleterious effect was detected on HIV-suppressive efficacy of ART. Surprisingly, our data suggest that schistosomiasis-induced immunomodulatory effects might be beneficial for survival and retention in care. Future studies are warranted to confirm these findings. In the era of increasing access to ART in sub-Saharan Africa, the issue of schistosomiasis-HIV coinfection may have major consequences on the outcome of HIV treatment programs.Infections with HIV and blood flukes ( Schistosoma prevalence reaches up to 30% in HIV cohorts [Schistosoma and HIV mutually interfere on several levels, which may impact the course of the associated diseases [Ascaris lumbricoides-HIV coinfected population found higher levels of immune activation, HIV-RNA concentrations, and lower CD4 T-cell counts in individuals with Th-2 bias, as indicated by high A. lumbricoides fecal egg counts, eosinophilia, and IgE response, compared to patients with high A. lumbricoides fecal egg counts, low eosinophil count, and low IgE responses [Schistosoma-HIV coinfected patients after antischistosomal treatment [The geographic distributions of HIV/AIDS and schistosomiasis largely overlap in sub-Saharan Africa, where  cohorts –4. In sediseases ,6. As bodiseases . Schistodiseases ,9. Propediseases . In lineesponses . In a streatment , and simreatment ,14.Schistosoma coinfection on the course of HIV infection in people living with HIV (PLWH) under antiretroviral treatment (ART) [Schistosoma-HIV coinfected patients starting ART, the odds for immunologic treatment failure were four times higher and CD4 cell count increases were significantly lower compared to PLWH without concurrent schistosomiasis [Schistosoma prevalence and massive roll out of ART in HIV cohorts, Schistosoma-induced treatment failure would have major implications for ART programs.To our knowledge, only two studies have investigated the effect of nt (ART) ,16. EfraSchistosoma coinfection has a negative impact on the course of HIV infection in patients starting ART. To test the hypothesis, we aimed to assess the effect of Schistosoma coinfection on patients’ response to ART in terms of (i) loss to follow-up (LFU) or mortality; (ii) immunological failure; and (iii) virological failure. The study was conducted in a well characterized HIV cohort in a rural part of south-central Tanzania and employed a highly sensitive diagnostic approach for schistosomiasis.The working hypothesis of the current study was that Schistosoma infection. Patients with positive test results were treated with praziquantel .The Kilombero and Ulanga Antiretroviral Cohort (KIULARCO) received ethics approval from the institutional review board of the Ifakara Health Institute (IHI) and from the National Health Research Ethics Review Committee of the National Institute for Medical Research of Tanzania. All patients provided written informed consent at inclusion in the KIULARCO cohort and parents or guardians provided informed consent on behalf of all participants under the age of 18 years. We re-tested all circulating anodic antigen (CAA)-positive patients for This study was carried out in Ifakara, a primarily rural region of south-central Tanzania. Patients were recruited from KIULARCO, an ongoing, open, prospective observational HIV cohort of PLWH followed at the Chronic Disease Clinic in Ifakara (CDCI). Further details of the KIULARCO cohort are given elsewhere ,18. WithThe study was a retrospective analysis of cryopreserved plasma samples and of data collected within the existing HIV cohort. The study included all patients who were enrolled in KIULARCO, started ART between January 1, 2013 and April 1, 2015, were older than 15 years, not pregnant, had a CD4 count performed at a maximum of 90 days before to 1 week after ART-start, and had at least one pre-ART plasma sample stored (at a maximum of 30 days before ART-start).Schistosoma infection status of each study participant at the moment of ART initiation was retrospectively assessed by testing 50 μl of pre-ART cryopreserved plasma for the presence of CAA with a lateral flow (LF) test with SCAA20 dry format [Schistosoma mansoni and S. haematobium (the two schistosome species endemic in Tanzania) and is shed into the host circulation during active infection. After successful treatment with praziquantel, antigen levels decrease within days [The y format ,20. Negay format ,20. CAA hin days . In addihin days –23. Prevhin days .Because isolated eosinophil count in peripheral blood is not part of routine diagnostic procedures in the KIULARCO, we used MXD  as approximate value. Cut-off for elevated MXD value was set at 1,100 cells/μl .The association between CAA-positivity and virological failure was assessed in a subgroup of study participants. Patients who were continuously CAA-positive  were frequency-matched with two controls (CAA-negative pre-ART and 6–13 months after ART-initiation) for age, CD4 cell count, and tuberculosis status at baseline. In these patients, plasma HIV RNA levels were tested in a cryopreserved plasma sample drawn 6–13 months after ART-initiation.TM Real-Time PCR System , with a detection limit of 60 viral RNA copies/ml of plasma. CD4 counts are routinely obtained after staining fresh whole blood samples with labeled antibodies: CD4, CD3, CD8, and CD45 in TruCount tubes .Plasma HIV RNA from 400 μl plasma was extracted using the NucleoSpin Virus kit  according to the manufacturer’s protocol. Viral RNA quantification was performed with the Brilliant III Ultra-Fast QRT-PCR Master Mix  using the StepOneData were extracted from a readily available electronic database of KIULARCO. The baseline was defined as the date of ART initiation. Continuous variables were summarized with medians and interquartile ranges (IQR) and categorical variables with frequencies and percentages.Association of CAA-positivity at ART-initiation with death or LFU was assessed with a multivariate Cox regression model. LFU was defined as no visit to the outpatient clinic for more than 6 months. The time of the event was defined as the day of the last follow-up visit documented in the database. Results were presented with hazard ratios (HR) and 95% confidence intervals (CIs). The assumption of proportional hazards was confirmed by Schoenfeld’s global test (p = 0.08).The association of CAA-positivity at ART initiation with immunological failure was assessed with a multivariate logistic regression model. Immunological failure was defined as CD4 count falling below baseline or persistently <100 cells/μl at the time of the first measurement of CD4 cell count ≥6 months after ART-start. Results were presented with odds ratios (ORs) and 95% CIs.a priori as potential co-factors/confounders and were included in the multivariable model (no variable selection was done): age (divided by 10), body mass index (BMI), and CD4 count at baseline (divided by 25) were used as continuous variables. Categorical variable included sex, educational attainment (none and primary school vs. secondary school and college/university), and WHO clinical stages of HIV disease (stage 1 and 2 vs. stage 3 and 4). The variable “active tuberculosis” was defined as diagnosis of tuberculosis at baseline or during follow-up. The variable “MXD value” was dichotomized in normal vs. elevated . The variable “year of starting ART” was added to the model used to assess the association of CAA-positivity with death/LFU. The variable “delay to CD4 testing”  was added to the model used to assess the association of CAA-positivity with immunological failure. Linearity of the relationship between continuous explanatory variables and the log odds of the dependent variable was tested by adding polynomial terms of each continuous explanatory variable. Polynomial terms showing a significant association with the log odds of the dependent variable were included in the model.For both models the following variables were considered For the analysis of CAA-positivity as a risk factor for virological failure , a logistic regression model was employed, controlling for the following potential co-factors/confounders (defined as above): sex, BMI, educational attainment, WHO clinical stages of HIV disease, MXD value, and delay to HIV RNA testing. Results were presented with ORs and 95% CIs.Data were anonymized and analyzed using STATA version 12.1 .2 , and median CD4 count was 173 cells/μl . CAA-positive patients had higher CD4 counts, higher MXD values, and there was a lower proportion of females  were CAA-positive. The median CAA titre at ART initiation was 163 pg/ml . After a median time of 36 weeks on ART , 36 remained CAA-positive, 17 became CAA-negative, and 35 had no follow-up plasma sample. In the group of 373 initially CAA-negative patients, 214 patients were continuously CAA-negative at ART-initiation and in the follow-up testing after a median time of 35 weeks , four became CAA-positive, and 155 had no follow-up plasma sample.Of 88 CAA-positive patients at ART initiation, 20 (22.7%) died or were LFU, compared to 110 out of 373 (29.5%) of CAA-negative patients . The median survival time in CAA-positive patients was 20.1 months , compared with 19.8 months  for CAA-negative patients.Baseline risk factors for death or LFU are presented in A follow-up CD4 count was available for 69 out of 88 (78.4%) of CAA-positive and for 287 out of 373 (76.9%) of CAA-negative patients. CAA-positive patients showed a median difference from baseline in CD4 of +182 cells/μl , compared to +147 cells/μl  in CAA-negative patients. Median time from ART initiation to measurement of CD4 count was 37.9 weeks  for CAA-positive patients, compared to 37.9 weeks  in CAA-negative patients. MXD values had no effect on CD4 count changes .Sixty-seven patients (18.8%) met at least one WHO criterion for immunological failure. CAA positivity showed no effect on the risk of immunological failure . Baseline risk factors for immunological failure are presented in One continuously CAA-positive patient and three continuously CAA-negative patients were excluded from the analysis due to non-availability of plasma samples for HIV PCR. After a median time of 43.4 weeks  of ART, eight out of 35 (22.9%) persistent CAA-positive patients had HIV RNA concentrations above 1,000 cp/ml, compared to 12 out of 67 (17.9%) of continuously CAA-negative patients after frequency matching for tuberculosis-status, CD4 count, and age. After adjustment for baseline risk factors, positive CAA status showed no association with virological failure [Schistosoma-HIV coinfection falsely negative tested Schistosoma coinfected patients would lead to misclassification and consequently to an underestimation of the effect under investigation. In such circumstances, the use of highly sensitive tests should be considered.The high prevalence of concomitant active schistosomiasis in this study, as determined by a highly sensitive assay  and of schistosomal and helminth infection in general in other studies in PLWH corroborate the potential importance of helminth-HIV coinfection ,16. Our hed data). We do nhed data),26,27. Thed data)–30. FromSchistosoma coinfection with an elevated risk for immunological failure or decelerated CD4 count gain in PLWH starting ART [S. mansoni, compared to controls without Schistosoma infection [In contrast to the findings of Efraim and colleagues, our study did not show an association of ting ART . Considenfection . Our resnfection –33.The rate of virological failure was substantial, which is a frequent problem, especially in rural settings of sub-Saharan Africa –36. EsseSchistosoma spp., which may exert beneficial effects in the setting of ART-induced immunological reconstitution. Immune reconstitution inflammatory syndrome (IRIS) is a condition seen in PLWH with profound CD4 T cell depletion and is characterized by an overwhelming inflammatory response to pathogens due to a recovering T cell count after initiation of ART. IRIS is a common cause of early mortality of patients starting ART in sub-Saharan Africa [Schistosoma-induced immunomodulation may have an attenuating effect on IRIS-related morbidity and mortality and by this means, improve the clinical outcome of PLWH starting ART.The protective effect of schistosomiasis in PLWH starting ART in our study is counterintuitive. Possible explanations of the results include immunomodulatory properties of n Africa . SchistoThe statistically significant association of elevated MXD values with a reduced risk of death and LFU may support this assumption. MXD value is composed of absolute cell counts of monocytes, eosinophilic, and basophilic granulocytes, which are independent from HIV infection and disease status –41. In tSchistosoma infection had elevated MXD values.Preceding studies in KIULARCO identified an important burden and variety of helminthic infections. Because immunomodulatory effects are a consistent feature of helminth infection, we believe that the weaker effect of schistosomal infection on survival/retention in care in our study may possibly be explained by competing immunomodulatory effects of other undetected helminths as our diagnostics in this study were focused on schistosomiasis. Indeed, about a quarter of PLWH without Our study has several shortcomings that are offered for discussion. First, clinical data of KIULARCO are collected prospectively, but CAA was identified retrospectively in cryopreserved plasma samples with the immanent weaknesses of a retrospective study design. Second, due to a suboptimal ascertainment of mortality in our cohort, we employed a composite outcome of death/LFU, limiting our conclusions regarding the impact of schistosomiasis on mortality. However, LFU is a common issue in ART programs in resource-limited settings and mortality is inversely associated with the rate of LFU . On the Schistosoma coinfection on immunological and virological response to ART but suggests that helminth-induced immunomodulatory mechanisms might enhance survival of PLWH starting ART.In conclusion, testing for CAA in plasma revealed a 19.1% prevalence of active schistosomiasis in PLWH starting ART in the KIULARCO in rural south-central Tanzania. This observation corroborates concerns of limited sensitivity of microscopy-based diagnostics, which might be additionally compromised in HIV-infected populations and may impede treatment for patients with a potentially life-threatening disease . Our stuOur results need confirmation in future studies, which should consider (i) the dynamic aspect of HIV infection and therefore preferentially chose a longitudinal study design, including clinical outcomes; and (ii) that individuals are often coinfected by multiple parasitic worms that may affect the course of the HIV infection by comparable but interindividually variable immunomodulatory effects. To avoid misclassification, comprehensive, highly sensitive helminth diagnostics and markers of the host’s immunological response should be employed. Among these highly sensitive tests, the used CAA-test is a promising candidate and further use and developments may improve the acceptance as a future standard test.In the era of massive rollout of ART in sub-Saharan Africa and other tropical and sub-tropical countries, the issue of helminth-HIV coinfection may have major ramifications on the outcome of HIV treatment programs. Further research, especially in PLWH initiating ART, is urgently needed.S1 STROBE Checklist(DOC)Click here for additional data file.S1 Dataset(DTA)Click here for additional data file.


Patient registries play an important role in obtaining real-world evidence of the cost-effectiveness of treatments. However, their implementation is costly and sometimes infeasible in many middle-income countries (MICs). We explored the combination of data-mining and a large claims database to estimate the direct healthcare costs of HER2-positive breast cancer (BC) treatment in Iran and the fraction of total costs from trastuzumab use.We performed a retrospective analysis of claims data from the Iran Social Security Organization, a health insurer which covers approximately 50%(~40 million) of the Iranian population, in the period of 21/03/2011-20/03/2014. A data-mining algorithm using R software and validated using patient dossiers in the Cancer Research Center identified 1295 patients and divided them into the three main HER2-positive breast cancer stages . A payer perspective was used to calculate the absolute and relative direct costs of healthcare services associated with the treatment of HER2-positive breast cancer in the public and private healthcare systems.The number of women totaled 802 (early), 125  and 218 (advanced). The mean age[SD] was 45, 4610]  10] and Wherever comprehensive patient registries are infeasible or costly, real-world costs can be estimated through claims databases and data-mining strategies. Using this method, real-world costs have been estimated in Iran. The stage-specific cost estimates derived from this study can be used to perform real-world cost-effectiveness analyses of therapies for HER2-positive BC and support healthcare financing decisions. Useful cost-effectiveness analyses require sufficiently detailed data on both costs and health outcomes to support decisions about the reimbursement or implementation of new interventions . PatientBC has become one of the most frequent malignancies among Iranian women , which hData from a claims database of the largest health insurance database in Iran were processed using data-mining, combined with data from other sources, and then analyzed to estimate the direct healthcare costs of HER2-positive BC in each disease stage from a payer’s perspective. The results of the data-mining process were verified using patient level-data. To access the above data sources, a research proposal was sent to the Social Security Organization (SSO) and the Cancer Research Center (CRC) to obtain claims data and patient-level data, respectively. After approval, we received the data requested in our proposal. The following sections describe the main parts of the study. Three sources were used to estimate healthcare resource use and costs. To start with, most data on outpatient services and costs were drawn from a major claims database of the Social Security Organization (SSO), a social and health insurance organization that covers more than 50% (~40 million insurees) of the whole Iranian population . Second,This step comprised several sub-steps which are as follows:The results of a survey of oncologists  and otheSince different types of data were needed to classify patients and estimate stage-specific costs, we prepared a list of necessary data in each category and then defined our data extraction strategy.Four filters were used to select patients with HER2-positive BC in the main SSO database: diagnosis of cancer, female, history of a mammography and trastuzumab use in the past two years. The Iranian national health services coding system was used to identify women who underwent a mammography and received trastuzumab. After identifying patients, all cost data associated with BC in various healthcare services  were then extracted from different databases.This step involved a check for duplications, completeness of data, and consistency of chemotherapy approaches. Any duplicated records were removed from the database. Then, to improve completeness and uniformity of data, two exclusion criteria were applied. Firstly, we excluded patients who had too few (n<6) “healthcare request sheets” over a period longer than two months since the date of the first prescription containing chemotherapy. It is worth nothing that these healthcare request sheets are used by doctors to prescribe necessary healthcare services for their patients. The Persian term for these sheets is “nos’kheh”, which we have translated to “healthcare request sheet” for lack of a better word. Each healthcare request sheet may contain one or multiple services. This is an instrument to connect doctors, other health care providers, and payers. We excluded patients with 5 or fewer healthcare request sheets because they had probably not yet completed the entire treatment regimen (type II right censoring). That is, patients who have completed the entire regimen should have at least six healthcare request sheets, which would consist of 3–4 healthcare request sheets for chemotherapy, 1–2 sheets for other medications such as hormone therapy, one sheet for laboratory test and one sheet for medical imaging during first two months of treatment onset. In fact, this threshold of 5 healthcare request sheets lies well below the number of prescriptions associated with an entire regimen. Secondly, patients were also excluded if they had a treatment regimen that was different than our previously defined treatment strategies.Classification algorithm was used to categorize patients into three different stages  based on medication patterns over a 3-year observation period (21.3.2011–20.3.2014). A decision tree induction using a divide-and-conquer approach  was usedIn general, data-mining resulted in the classification of patients into three health states, including a patient-specific time interval representing the start and end dates of treatments at any particular health state.Patient-level data from the CRC were used to verify the results of the patient classification algorithm. In total, there were 51 patients at this center who were HER2-positive and SSO beneficiaries; HER2-positive BC was based on whether an IHC result showed two or more pluses or the FISH test was positive. Verification involved comparing the results of the algorithm results with the clinical results based on the tumor, node, and metastases (TNM) classification system. We examined if patients categorized as having early stage BC by the classification algorithm were in stages I or II of BC, if patients with loco-regional BC had stage III BC and if patients with advanced BC had stage IV BC. The SSO’s ID of patients in the CRC were used to link this small dataset to the claims database by SSO’s database management staff. The result of any patient classification by data-mining was approved (based on expert opinion), firstly, if the same stage had been reported in the patient dossier. Secondly, if the stage-specific index date obtained from data-mining was within two months greater than the stage-specific diagnosis date in the patient dossier.A national coding system covers both inpatient and outpatient healthcare services in Iran. Each healthcare service (e.g. a tablet of acetaminophen or open-heart operation) has a unique code. The national coding system is developed by the Ministry of Health. Healthcare providers must use this system (drugs + other services) when they seek payment for the procedures and services they have provided to individual patients. Healthcare providers send a text file including the patient’s ID, national codes of all services used for that patient, doctor’s ID and costs (co-payments plus payers’ share costs) to the SSO , either daily or monthly. Ultimately, knowing the code of any services make it possible to identify users, prescribers and the costs of that particular healthcare service.Considering the above information, firstly, ‘service-specific costs’ were estimated using the national coding system. Outpatient care costs were calculated based on prevalent healthcare services . Inpatient costs were estimated by extracting data from two hospitals on the surgical procedures used in BC management ; this data were extracted from the same period as the outpatient data (21.3.2011–20.3.2014).Afterwards, we used the results of data-mining to calculate ‘stage-specific costs’. As mentioned earlier, data-mining was used to categorize patients into three health states. Moreover, data-mining helped us to determine the duration of time that individual patients spent in each health state . Therefore, stage-specific costs over time were estimated as follows. Costs of various outpatient services  were calculated based on the individual stage-specific time interval. For those patients whose outpatient and inpatient data were available, inpatient costs were categorized based on their stage-specific time interval obtained from data-mining on outpatient data.In both service- and stage-specific costs, we also included treatment costs in the private sector, since the unit costs can differ between the public and private sector and patients are free to choose their healthcare provider ,13. TherThe claims data were analyzed to determine the mean and distribution of resource use and costs of different medical treatment regimens, including chemotherapy, hormone therapy, radiotherapy, laboratory tests, medical imaging services and surgery in the different BC stages. 95% confidence intervals (CIs) were calculated using non-parametric bootstrapping. The number of iterations was 2000 times in all cases. All data-mining process and calculations were made using R software (version 3.2.2) for Windows .The data extraction process yielded 1,295 patients with an average age [SD] of 44  years T. 54 patiVerification of patient classification was based on data obtained from CRC on a group of 51 patients; 65% (n = 33) were in early BC, 10%(n = 5) in loco-regional BC and 25%(n = 13) in advanced BC. Two (4%) patients had both loco-regional and advanced BC in the observation period. Some patients with loco-regional and advanced BC were incorrectly categorized as having early stage BC; these represented 14% (n = 7) of patients in the loco-regional stage and 2% (n = 1) of patients with advanced BC). Therefore, the accuracy by stage was 100% (25/25) for early BC, 46% (6/13) for loco-regional BC and 93% (14/15) for advanced BC. In general, verification of the classifications based on data-mining revealed an 85% accuracy rate.The different outpatient costs are shown in In all three BC stages, there is a rapid increase in outpatient-related costs during the first year after the index date , followeAs The average cumulative direct healthcare costs for early BC over the follow-up period were €11,796 . The treatment costs for loco-regional BC averaged €8,253 . In contrast, the mean costs for advanced BC (95% CI) were €17,742 , which was roughly 50% higher than the costs for early stage BC.Generally, BC is a costly illness throughout the world. The use of monoclonal antibodies like trastuzumab or pertuzumab to treat BC can have a significant budget impact, specifically in MICs. We performed a real-world cost analysis using a large claims database and a validated patient classification algorithm to estimate the stage-specific healthcare utilization and costs associated with HER2-positive BC in Iran.Our findings show that total direct healthcare costs per patient averaged €11,796 for early BC, €8,253 for loco-regional BC and €17,742 for advanced BC during a 3-year follow-up period (21.3.2011–20.3.2014). The largest share of the total cost was spent on trastuzumab. Other medications like chemotherapy and hormone therapy were the second most important cost drivers in BC. The relative costs of other medications associated with advanced BC were dramatically higher compared with early and loco-regional BC . This waA previous study estimated the direct costs of treating patients with BC based on patients referred to one hospital in Iran . AlthougLike any study, our study also has its limitations. The first limitation relates to our study population. One could argue that the claim data covers approximately 50% of the population and therefore may not represent the whole Iranian population. However, our results can be generalizable to the whole population because the four main payers in Iran have the same benefit package and because the SSO, which provided the data used in our study, covers the insurees from different socio-economic classes in all provinces . A seconThis study shows that if relevant data are available, data-mining techniques on claims data can provide cost estimates that are invaluable in performing real-world cost-effectiveness analyses in MICs. National level cost-effectiveness analyses can use these cost estimates to assess whether new medicines or procedures are cost-effective. For example, policymakers can reduce uncertainties about the cost-effectiveness of a monoclonal antibody like trastuzumab by performing a country-specific cost-effectiveness analysis using the results of our real-world cost analysis. Despite the advantages of using claims databases, these data sources usually do not provide clinical, laboratory, and imaging data as well as some important treatment events such drug-related adverse events.The quality of reimbursement decision-making can be improved by using information about actual healthcare utilization and costs. One option in high-income countries would be an extensive cost analysis supported by patient registry data and electronic health records. However, implementation of comprehensive patient registries is costly and sometimes logistically infeasible in many MICs. If integrated electronic records are unavailable, researchers may have to rely on small sample sizes or expert opinion  t) t24]) tThis study estimated the stage-specific direct healthcare costs associated with HER2-positive BC in Iran using a large claims database and data-mining with validated patient classification algorithms. The findings show that the largest component in overall costs in all stages of BC is medication and that trastuzumab is the major cost driver. These real-world data can support cost-effectiveness analyses of implementing new technologies for HER2-positive BC management and thereby help to optimize reimbursement decision-making in an MIC.