Loa loa is a filarial species found exclusively in West and Central Africa. Microscopy is the traditional diagnosis method for human loiasis. Several molecular methods have developed as an alternative approach for identification of L. loa filarial parasites. The aim of this study was to evaluate a Loa-Loop-mediated isothermal amplification (LAMP) assay to diagnose loiasis disease on dried blood spots (DBS) samples, compared to microscopy, filaria-real time-polymerase chain reaction (PCR) and nested-Loa PCR. A total of 100 DBS samples and 100 blood smears were used for this study. DNA was extracted using saponin/Chelex method. DNA isolated was assayed by a Loa-LAMP assay in parallel to microscopy, filaria-real time PCR and nested-Loa PCR. The sensitivities and specificities of Loa-LAMP assay was computed comparing to each one of the reference methods. Loa-LAMP’s sensitivity was more than 90% and specificity was nearly 100% when compared to molecular methods. On the other hand, sensitivity was decreased a bit when Loa-LAMP faced microscopy, but keeping the other statistical values high. Loa-LAMP is an appropriate method for loiasis diagnosis in endemic areas. Though, it has disadvantages like the reagents’ high price at the moment and not to be able to detect more filarial species at once. These parasites are nematodes (roundworms) and are found in different adult tissues, depending on the species.Loa loa is a human filarial species, found exclusively in Western and Central Africa and transmitted by the bite of the Chrysops deerfly. This filarial worm causes loiasis, also known as African eye worm.L. loa co-exists with Onchocerca volvulus, Wuchereria bancrofti and Mansonella perstans.,,,L. loa was traditionally an absent filaria for the WHO, it began to receive increasing attention when severe adverse events presenting as encephalopathy occurred in onchocerciasis control projects supported by the African Programme for Onchocerciasis Control following treatment with ivermectin. Hence, in areas where loiasis is co-endemic with onchocerciasis and/or LF, the ability to safely carry out mass drug administration with ivermectin is impeded, especially at very high microfilarial loads ).,L. loa in onchocerciasis and/or LF co-infections, this filarial parasite is not included in the WHO’s list of NTDs. However, the Expanded Special Project for Elimination of Neglected Tropical Diseases (ESPEN), established in May 2016, which is a project dedicated to the control and elimination of the five NTDs amenable to preventive chemotherapy  does include L. loa in the list of diseases to be eliminated.Although L. loa mf are long and thick (205-300 x 6-8 µm),with a sheath and a terminal nucleus in their tail.,,,,,Conventionally, laboratory diagnosis of human loiasis relies on the detection of mf on Giemsa-stained or haematoxylin/eosin-stained thick and/or thin blood films by microscopy, with or without prior concentration.,,,Several polymerase chain reaction (PCR) techniques have been developed to detect Loa-filarial infection in humans, with a high accuracy for detecting single or mixed infections with other filarias. As such, they have been shown to be an excellent alternative diagnostic method to microscopy. However, PCR methods have the disadvantage of requiring expensive equipment, laboratories with good infrastructure, a reliable electrical supply and highly trained staff, in addition to a long amplification process.L. loa pathogen has also been developed to overcome the limitations of the PCR techniques. This molecular method is relatively recent, having emerged in 2000,Loop-mediated isothermal amplification (LAMP) for ,As such, the LAMP assay has emerged as a new molecular method with the potential to replace PCR methods in the near future given its numerous advantages and outstanding efficiency.This study has been performed as the National Centre of Tropical Medicine  carries out international scientific-technical cooperation programs with African countries where this disease is found. Recently, the Centre was awarded several projects to assess human filariasis transmission in Equatorial Guinea and develop national recommendations to move towards onchocerciasis elimination and LF/loiasis control at a country level.The aim of this study was to evaluate the usefulness of a previously described LAMP assay to diagnose loiasis disease in stored samples, preserved as dried blood spot (DBS), compared to the reference methods used in the laboratory for the diagnosis of loiasis disease, namely microscopy, filaria-real time-PCR and nested-loa PCR.Clinical samples - DBS and thick and thin blood smear (on the same slide) samples were obtained from the Laboratory of the National Centre of Tropical Medicine’s repository (collection number C.0005278/ISCIII/Spain), which is registered according to the Spanish Law RD 1716/2011 (article 22. 1). Both DBS and thick and thin blood smears belonged to the malaria project PREVAMAL (2013), to provide baseline data on malaria prevalence in Equatorial Guinea.Microscopy - The thick and thin blood smears were used to characterise the mf and calculate the microfilaraemia taking into account that each thick and thin blood smear contained approximately 20 and 5 µL, respectively. Morphological identification for mf was performed in line with published guidelinesDBS - Positive and negative controls were prepared using a fresh whole blood sample in EDTA, supplied by the Malaria and Emerging Parasitic Diseases Laboratory . Thus 70 µL of blood was spotted onto Whatman filter paper and the spot allowed to dry in air for one day. DBS from infected and uninfected persons was previously diagnosed by microscopy on a blood smear. For each set of extractions performed, positive and negative DBS samples, as well as a blank (filter paper without blood), were always executed in parallel with the undiagnosed samples.DNA extraction - DNA was extracted from DBS using the classical saponin/Chelex method, with slight modifications from the original protocol.Filter paper samples were handled according to safety procedures to prevent contamination of samples with each other by disinfecting the handle punch in NaOH 5 M solution and rinsing the residual NaOH with distilled water.2+ ions.Prior to molecular testing, the isolated DNA samples should be centrifuged at 13,000 rpm for 2 min to place the remaining Chelex at the bottom of the tube. Chelex-100 is a chelating resin with a high affinity for multivalent metal ions, so it can inhibit the PCR by binding to Mg,The quality of the DNA obtained using the saponin/Chelex extraction method was checked using Nested-Malaria PCR method, which uses the human small subunit ribosomal RNA gene sequences as internal control.Molecular methods - The presence of L. loa parasites in the DNA isolated was subsequently assayed using three different molecular methods, targeting different genes to diagnose loiasis infections, as indicated briefly below:® Universal qPCR Master Mix 2x (New England Biolabs). The cycling conditions comprised an initial denaturation step at 95ºC for 1 min, followed by 45 cycles of denaturation at 95ºC for 15 s, annealing at 48ºC for 20 s and extension at 60ºC for 30 s. A sample was considered positive for filarial parasite if the melting temperature (Tm) curve of the amplified fragments was Tm =77ºC ± 0.5ºC and the species identification was according to amplified product size after analysing in a multicapillary electrophoresis.- Filaria-real time-PCR (F-RT-PCR): targeting the internal transcribed spacer one (ITS1) of the nuclear ribosomal gene of all filarial species.L. loa 15-kD protein.- Nested-Loa PCR: this nested PCR targets the gene encoding the L. loa-specific gene sequences LLMF72.Bst DNA polymerase. Positive amplification results were detected by the naked eye based on the colour change using the pH-sensitive dye Phenol red.- Loa-LAMP: the isothermal amplification assay was designed to target the L. loa-microscopy positive individuals; negative controls were DBS samples of L. loa-microscopy negative individuals from filarial-endemic regions who were negative for filarial infection but could be positive or negative for other tropical parasites, such as Plasmodium spp.For each reaction performed, appropriate positive and negative controls were always included. Positive controls were DBS samples of All samples were run in duplicate. If the duplicates varied , the samples were rerun in triplicate.®PCR System 2700. Amplification products were detected using QIAxcel Advanced , an automated system for analysing DNA fragments based on multicapillary electrophoresis.All F-RT-PCR reactions were performed using a Rotor Gene Q 5plex . The remaining DNA amplifications, including the Loa-LAMP, were performed using an Applied Biosystems GeneAmpSee DNA sequencing and sequence analysis - All the products amplified by F-RT-PCR and Nested-Loa PCR techniques were purified using Speedtools PCR Clean Up Kit 250 rxns , then sequenced in both directions using Big Dye Terminator v3.1 Cycle Sequencing in an ABI PRISM® 3700 DNA Analyzer. Sequences were compared to the genbank database using basic local alignment search tool (BLAST)In the case of the Loa-LAMP method, the positive post-LAMP reaction mixture subjected to gel electrophoresis produces many bands of different sizes in a reproducible ladder-like pattern and these products could not be sequenced.Statistical analysis - Statistical values  for the Loa-LAMP assay was calculated compared to each of the reference methods using the free software WinEpi: Working in Epidemiology.Overall, 66 samples were identified as negative by all the tests and 34 samples were positive in at least one of the methods tested (microscopy or molecular test). A list of positive samples has been created to facilitate the following and understanding of the results .Microscopy - Out of 100 thick and thin blood smears analysed by microscopy, 73 (73%) were negative and 27 (27%) were positive: 13 were L. loa, 11 M. perstans and 3 were mixed infections (L. loa + M. perstans). The average microfilaremia for L. loa was 3047 mf/mL (parasite count range 200-12200 mf/mL) and for M. perstans was 764 mf/mL (parasite count range 100-3200 mf/mL) (0 mf/mL) .F-RT-PCR - This method was able to detect 30 (30%) positive samples: 11 L. loa, 17 M. perstans and two mixed infections. Seven M. perstans-F-RT-PCR positive samples were missed by microscopy, whereas four microscopy positive samples were missed by F-RT-PCR  (fection) .Nested-Loa PCR - This method detected 12 (12%) L. loa positives and 88 (88%) L. loa negatives (L. loa-microscopy positives (3 L. loa single and 1 mixed infection) were missed by Nested-Loa PCR.egatives . Four saLoa-LAMP - This method detected 13 (13%) L. loa positives and 87 (87%) L. loa negatives, and missed the same four samples as Nested-Loa PCR . The concordance (kappa index) was good or excellent. On the other hand, sensitivity (75%) was slightly lower when comparing Loa-LAMP with microscopy, although the other statistical values remained high . Blood samples with very low microfilaremia have a lower possibility of taking one microfilariae in the punch because the distribution of mf on the blood spot is not homogenous. Thus, parasite material seems to be less concentrated towards the extreme edges of the blood spot, as shown in the experiment carried out by Baidjoe et al.L. loa + M. perstans) detected by microscopy, no loa parasite could be detected in one sample by any of the three molecular methods. This mixed infection also had a very low microfilaremia (200 mf/mL).The samples that were ,False-negative results due to the presence of potential enzyme inhibitors in the samples, or the poor DNA quality or concentration yielded by the saponin/Chelex DNA extraction method, were discarded, since all of them were checked with an internal extraction control present in the Nested-Malaria PCR method.M. perstans were also included in this study to identify possible cross-reactivity with M. perstans using the Loa-LAMP. There was only one case (#137) that tested M. perstans-F-RT-PCR positive and positive for L. loa by Loa-LAMP. This could be a possible mixed infection in which each method was able to detect the filarial parasite for which it is most sensitive. Overall, Loa-LAMP did not exhibit a cross-reaction with M. perstans DNA, although the possibility of a cross-reaction cannot be excluded.Samples infected by M. perstans-microscopy positive (#79) was missed by F-RT-PCR, probably because this sample had 200 mf/mL. Again, the sensitivity of the molecular method appeared to be compromised because of the low microfilaremia and the lower probability of collecting the microfilariae. It should be noted that F-RT-PCR detected seven more M. perstans which were not detected by microscopy.Only one After evaluating the Loa-LAMP assay, it has been found that the sensitivity and specificity are very high and the concordance between methods shows excellent agreement according to the kappa index values, thus suggesting that the method may be suitable for use with clinical samples in resource-limited areas endemic for loiasis.L. loa DNA in the field. Indeed, its statistical values are very similar to those found for F-RT-PCR and Nested-Loa PCR when tested with the same clinical samples.As can be seen by the results, the Loa-LAMP assay may also be very useful as a potential point-of-care tool for the rapid amplification and easy detection of Despite using a different source of samples, another method for DNA isolation and different reagents, the results obtained from the colorimetric LAMP assays in this study are similar to those reported by Drame et al.,In our opinion, the major limitation of the LAMP assay is the price of the kit used. Thus, in our study, the cost per sample was 3.5-times higher than for real-time PCR and almost five-times higher than for Nested-Loa PCR. As such, this could be a major limitation for its use in developing countries. In addition, the Loa-LAMP method still needs electrical power for the water bath or heating block to carry out the reaction, and a freezer is needed to store the Loa-LAMP reagents, and not all the endemic areas have a continuous power supply.L. loa DNA even though an absence of L. loa DNA does not rule out the presence of other blood microfilariae (M. perstans or W. bancrofti).Another disadvantage of this Loa-LAMP technique is that it is monoplex and, therefore, has a limited capacity to detect a wide variety of filarial species at once. Indeed, it has been specifically designed to detect In conclusion - As illustrated by the results from this study, Loa-LAMP is very similar in terms of sensitivity and specificity to established diagnostic methods for detecting loiasis infection, such as microscopy, F-RT-PCR and Nested-Loa PCR. As such, it is an appropriate method for loiasis diagnosis if a rapid screening of patients in endemic areas is required. Moreover, Loa-LAMP is a good alternative for use in the field, as no major capital equipment is required, the turnaround times are short compared to F-RT-PCR and Nested-Loa PCR, and amplification by colorimetry can be detected by the naked eye, although the main disadvantages are the high price of reagents and the inability to detect more than one filarial species simultaneously. Given the good results for the diagnosis of LAMP, its applicability in the field and the ease with which positives can be detected, laboratories such as ours should design a LAMP that can detect more than one species simultaneously in order to facilitate work in the field. The basis of good scientific/technical cooperation, one of the responsibilities of our Centre, is to be able to offer endemic areas fast and reliable diagnostic tools that facilitate diagnosis, so that patients can quickly receive effective treatment.Ethics approval and consent to participate - All the clinical samples, DBS and blood smears, had the authorisation of the director of National Centre of Tropical Medicine to be used in this study with the unique purpose to compare the Loa-LAMP assay to microcopy, and two molecular techniques, filaria-real time-PCR and nested-loa PCR.List of abbreviationsBLAST: Basic Local Alignment Search Tool; bp: basepairs; ºC: degrees Celsius; CI: confidence intervals; DBS: dried blood spots; DNA: deoxyribonucleic acid; EDTA: ethylene diamine tetra-acetic acid; ESPEN: Expanded Special Project for Elimination of Neglected Tropical Diseases; ITS1: internal transcribed spacer one; LAMP: Loop-mediated isothermal amplification; LF: lymphatic filariasis; F-RT-PCR: filaria-real time-PCR; mf: microfilariae; mL: milliliter; mm: millimeter; min: minutes; NPV: negative predictive value; NTDs: neglected tropical diseases; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PPV: positive predictive value; rDNA: ribosomal deoxyribonucleic acid; RNA: ribonucleic acid; rpm: revolutions per minute; WHO: World Health Organization; s: seconds; Tm: melting temperature; µL: microliter.


Like all cells, hematopoietic stem cells (HSCs) and their offspring, the hematopoietic progenitor cells (HPCs), are highly sociable. Their capacity to interact with bone marrow niche cells and respond to environmental cytokines orchestrates the generation of the different types of blood and immune cells. The starting point for engineering hematopoiesis ex vivo is the nature of HSCs, and a longstanding premise is that they are a homogeneous population of cells. However, recent findings have shown that adult bone marrow HSCs are really a mixture of cells, with many having lineage affiliations. A second key consideration is: Do HSCs “choose” a lineage in a random and cell-intrinsic manner, or are they instructed by cytokines? Since their discovery, the hematopoietic cytokines have been viewed as survival and proliferation factors for lineage committed HPCs. Some are now known to also instruct cell lineage choice. These fundamental changes to our understanding of hematopoiesis are important for placing niche support in the right context and for fabricating an ex vivo environment to support HSC development. HSCs produce a wide range of cell types, each with unique functions, that include platelets, erythrocytes, basophils/mast cells, eosinophils, neutrophils, dendritic cells, B lymphocytes, innate lymphoid cells, and T lymphocytes. For some of these major cell lineages, for example, dendritic cells, B lymphocytes, innate lymphoid cells, and T lymphocytes, there are sub-populations of cells with a more precise function. The main cell types, with the exception of the mature T lymphocytes, are generated in the adult by the bone marrow. An engineered marrow-like environment for HSCs should enable the extensive multipotent nature of HSCs. A further requirement is the need to simulate both the steady state production of the blood cell types and the increased production of a particular type of cell during emergency hematopoiesis. For example, there is an urgent need to generate, megakaryocytes giving rise to platelets in the case of thrombocytopenia, and neutrophils to control a bacterial infection.The responsiveness of cells to their environment is crucial to the cohesion of multicellular organisms and to meeting an organism’s changing demands. For the many types of cells, the environmental controls on cell survival versus cell death ensure proper cell numbers and also eliminate cells that are in the wrong place . Similar−Sca1+c-Kit+ (LSK) cells. Within this population, for example, LT-HSCs are LSK CD150+ CD48- CD34- and ST-HSCs are LSK CD150+ CD48− CD34+, whereby a gain of CD34 expression separates these populations  mouse  and neweulations A. A sing in mice . AccordiHowever, sub-populations of murine HSCs have been identified that selectively express receptors for some of the cytokines that are lineage-affiliated. As tested by transplantation experiments in mice, each sub-set also mainly gives rise to cells of one cell type. A sub-population of HSCs that requires thrombopoietin for maintenance, and that expresses the megakaryocyte-affiliated von Willebrand factor, gave rise to a long-term platelet- or platelet/myeloid-biased reconstitution when transplanted as single cells . The ide+ compartment of human adult bone marrow revealed mostly unipotent progenitors, with few oligo-potent progenitors. A shift occurs during the transition from in utero to adult hematopoiesis, because fetal liver contained large numbers of oligo-potent progenitors ..29].+ T-cell effector cells produce different cytokines relating to their functionality. The sub-sets include T helper 1 cells, T helper 2 cells (Th2), interleukin 17-producing T helper cells (Th17), follicular T helper cells (Tfh), and regulatory T cells (iTreg). Th2 cells can convert to Tfh [The developmental progression of HSCs is much more dynamic than a simple linear pathway towards an end cell type. There is flexibility to ensure that hematopoiesis is adaptable to the demand for a particular cell type(s). HSCs and HPCs that have “chosen” a cell lineage remain versatile as they can develop into a mature cell that does not belong to the initial choice. RNA sequencing of single mouse HSCs/HPCs revealed alternative trajectories . Megakart to Tfh , iTreg tt to Tfh , and memt to Tfh , and celt to Tfh .The existence of lineage-affiliated HSCs means that a distinction between HSCs and HPCs becomes somewhat redundant. Additionally, self-renewal, which has long been viewed as a cardinal property of HSCs, does not go strictly hand in hand with pluripotency. The repopulation competence of single lineage-affiliated HSCs indicates that they are likely to self-renew. In this scenario, fundamental questions about the fate of HSCs are: How do they “choose” a cell lineage, and do niches play a key role? Investigators have debated at length whether HSCs “choose” a cell lineage in a random and cell-intrinsic manner or whether they are instructed by cytokines, presumably presented by niche cells. Choosing either nature or nurture, respectively, is reductionist and overly simplistic. Longstanding and recent findings support the view that both of these influences drive HSCs to adopt a lineage fate.RUNX1 enhancer, is important for the emergence of HSCs [Studies of the multipotent murine hematopoietic cell line, EML, revealed that transcriptome-wide noise plays a role in controlling lineage choice. Cells that expressed the stem cell marker Sca1 at either an extremely high or low level had distinct transcriptomes, and their unique gene expression profiles fluctuated, eventually reverting to that of the median cells. The profile lasted long enough to confer a propensity towards development along either the erythroid or myeloid pathways. The conclusion was that the protein level fluctuations that render this proclivity are a manifestation of “gene expression noise” . Similar of HSCs .SATB Homeobox 1 (SATB1) gene encodes a matrix protein that binds nuclear matrix and scaffold-associating DNAs and SATB1 regulates chromatin structure and gene expression by means of recruiting chromatin-modelling factors. Variable levels of SATB1 correlate with the heterogeneity of HSCs regarding their distinct lineage fates [The underlying structure of chromatin is important for the translational noise within the levels of mRNAs, because this correlates with the three-dimensional organization of nuclear domains . The SATge fates . There ige fates , adding + mouse bone marrow cells lacking lineage surface markers. Both of these cell populations expressed cytokine receptor mRNAs at low levels. All of the FDCP-mix cells expressed the receptor for IL-3, in keeping with their IL-3-dependence, whereas a variable percentage of cells expressed the receptors for Epo (10%), M-CSF (22%), G-CSF (54%), and GM-CSF (48%). FDCP-mix cells also appeared to co-express receptors, leading investigators to the view that individual cells transit through the low-level priming of different lineage-affiliated loci [Early studies examined lineage priming within single HSCs and concluded that the expression of the genes that encode the receptors for the hematopoietic cytokines is fundamental to preparing for exclusive lineage-affiliation. Investigators made use of the sensitivity of RT-PCR to measure low levels of mRNAs for genes that are lineage-affiliated within FDCP-mix cells, derived from mouse bone marrow and that self-renew and undergo multi-myeloid differentiation, and CD34ted loci .The existence of HSCs that selectively express receptors for lineage-affiliated cytokines at a low level is highly germane to how HSCs “choose” a lineage fate. For many years M-CSF, G-CSF, GM-CSF, Epo, and Flt3L were viewed as survival and proliferation factors for lineage committed HPCs, but they are now also known to instruct cell lineage choice.+ cells have a myeloid signature [M-CSF instructs myeloid lineage fate within HSCs. Highly purified HSCs were treated with M-CSF in culture and the use of single-cell gene video imaging and expression analysis revealed that M-CSF induces the master myeloid transcription factor PU.1 and that an increased number of PU.1ignature . M-CSF aignature . Bio-imaignature .−. This change in the pattern of gene regulation was also observed post-treatment in LSK CD150+ Flt3− cells with Epo in vitro. Transplantation of Epo-exposed HSCs/multipotent HPCs into sub-lethally irradiated mice generated higher numbers of erythrocytes and fewer myeloid cells. Similarly, erythroid colonies were increased when a population of marrow cells from the mice that contained both myeloid and erythroid progenitors (LSK cells) was plated in conditions for both myeloid and erythroid differentiation. The conclusion from these studies was that the instructive action of Epo extends from HSCs to HPCs [Epo instructs erythroid fate within mouse HSCs/multipotent HPCs. Investigators used a CMV-based Epo expression vector to increase the systemic level of Epo in mice to that observed in anaemic patients. Erythroid commitment-associated genes were upregulated and megakaryocyte commitment- and granulocyte/macrophage commitment-associated genes were down-regulation within HSCs/multipotent HPCs that were purified from the mice as LSK Flt to HPCs . As mentThe above cytokines can regulate HSC/HPC survival, proliferation, and lineage commitment, but a high level of cytokine was required in the in vivo and ex vivo experimental studies to instruct cell lineage, as seen for the Flt3 ligand transgenic mice and the use of an Epo expression vector. Perhaps the level is important, whereby a low level is sufficient for survival, a raised level drives proliferation, and a further increase instructs cell lineage. It is interesting to speculate that the latter is more relevant for increasing the output of a cell type, rather than steady state hematopoiesis. In particular, lineage-affiliated HSCs enable an organism to meet an emergency requirement for a particular cell type(s). Following the acute depletion of platelets, investigators observed a rapid recruitment of platelet-biased (vWF+) HSCs, and perivascular niche cells sensed thrombocytopenia and activated platelet-biased HSCs to replenish circulating platelets rapidly . During + erythroid-affiliated HSCs need to pursue their terminal erythroid development program. Niche compartmentalization of the provision of cytokine-mediated signaling would mean that there is a need for HSC sub-populations to locate to different niches in the marrow. How might they do so? SCF is a chemo-attractant for human CD34+ HPCs from cord and peripheral blood. Grafting of human Nalm-6 pre-B acute lymphoblastic leukemia cells into SCID mice created malignant niches that secreted SCF, which, in turn, led to the migration of the human HPCs into the niches, including HPCs that were pre-established in marrow [The cytokine requirements of multipotent and lineage affiliated HSCs are likely to be different, for example, EpoRn marrow . That Epn marrow , endothen marrow , and humn marrow . M-CSF in marrow , monocytn marrow , macrophn marrow , and 32Dn marrow . GM-CSF n marrow , endothen marrow , and mesn marrow . We mighAn intriguing speculation is that HSCs that express one of the lineage-affiliated cytokine receptors at a low level migrate along a gradient towards a particular niche that is rich source of the cytokine, as presented by stromal cells . Whilst Future advances in the field of bone marrow tissue engineering, with the means to control niche components, will no doubt facilitate gaining a better understanding of how HSCs develop towards an end cell type. This information should offer the prospect of generating large numbers of specific types of hematopoietic cells for therapeutic purposes, including from HSCs and HPCs from human pluripotent stem cells  or inducAn understanding of how certain bone marrow niches control HSC development is clearly germane to resolving what occurs when the bone marrow is given over to producing an abundance of leukemia cells. Moreover, the stem cell theory of cancer, proposed from early studies of acute myeloid leukemia, states that most leukemias arise from HSCs . The leuUnlike the diverse progeny of normal multipotent stem cells, the bulk cells of the hundreds of distinct cancer types resemble just one mature or partially mature cell type, even when the origin is a multipotent stem cell. This is clearly seen for chronic myeloid leukemia (CML) and acute erythroid leukemia, which arise in an HSC with the progeny belonging to just the neutrophil and erythroid lineages, respectively. Some leukemias were thought to arise in a lineage-committed HPC, but further studies revealed they actually have an HSC origin. Infant B acute lymphoblastic leukemia cells are immature B-lymphoblasts, but genome-wide analysis identified a fetal liver HSC as the origin . Common Further evidence supporting the view that LSCs are lineage restricted is that some oncogenes impose a lineage fate on HSCs. Investigators made use of transgenic mouse models to investigate the initiation of leukemia in HSCs. The LMO2, BCR-ABLp190, and BCR-ABLp210 oncogenes play key roles in the onset of human acute T-lymphoblastic, pre-B acute lymphoblastic, and CML, respectively. Restriction of the expression of each oncogene to HSCs, via the Sca-1 promotor, allowed investigators to determine whether these oncogenes alter the behavior of HSCs. In the mice, each oncogene led to the respective human-like lineage-restricted leukemia  73,74,7,774,75. The behavior of leukemia cells, and cancer cells in general, is anarchic. They have escaped from the needs of the organism as a whole because they seem to be unable to follow a pathway that is different from that which is oncogene-wired. Even so, the survival of leukemia progenitor cells remains cytokine- and stromal cell-dependent. The survival of pre-B ALL cells ex vivo is supported by IL-3 and IL-7, and stromal cells . CML proStochastic modelling of lineage affiliation is highly suited to describing complex systems and HSCs as independent decision-makers, for self-renewal and cell lineage choice . The infper se in regenerative medicine.Fifty years ago, investigators observed, by electron microscopy, specialized niches for erythropoiesis, termed erythroblastic islands, that ensure autonomous and terminal-erythroid differentiation . However


Joint torques of lower extremity are important clinical indicators of gait capability. This parameter can be quantified via hybrid neuromusculoskeletal modelling that combines electromyography-driven modelling and static optimisation. The simulations rely on kinematics and external force measurements, for example, ground reaction forces (GRF) and the corresponding centres of pressure (COP), which are conventionally acquired using force plates. This bulky equipment, however, hinders gait analysis in real-world environments. While this portability issue could potentially be solved by estimating the parameters through machine learning, the effect of the estimation errors on joint torque prediction with biomechanical models remains to be investigated. This study first estimated GRF and COP through feedforward artificial neural networks, and then leveraged them to predict lower-limb sagittal joint torques via (i) inverse dynamics and (ii) hybrid modelling. The approach was evaluated on five healthy subjects, individually. The predicted torques were validated with the measured torques, showing that hip was the most sensitive whereas ankle was the most resistive to the GRF/COP estimates for both models, with average metrics values being 0.70 <  Three-dimensional gait analysis aimed at quantifying joint and musculotendon kinetics is crucial for certain medical diagnosis, motor assessment and development of rehabilitative technologies ,2,3. EstIn gait analysis, both inverse dynamics and the hybrid model rely on kinematics and external forces such as ground reaction force (GRF) and the corresponding centre of pressure (COP). Conventionally, these measurements are acquired using camera-based motion capture systems and force plates installed under the walkway, which as a concept poses some limitations. The size and weight of these instruments constitute the constraint of nonportability, causing real-world analyses to be almost infeasible. The high cost of the force plates further complicates analyses with a large number of consecutive gait cycles. To address the issues induced by force plates, past studies have attempted the use of pressure insoles and instrumented shoes ,26,27,28Some previous studies have directly predicted joint torques via data-driven methods without relying on GRF or COP ,39,40,41The primary aim of this study is to explore the applicability of both the inverse dynamics and the hybrid neuromusculoskeletal model for sagittal joint torque prediction in less constraining environments. This implies limiting the use of force plates and relying on measurements that can eventually be obtained with fully wearable systems. We first estimated GRF and COP with machine learning algorithms, then used these estimates to support the prediction of joint torques via inverse dynamics and hybrid neuromusculoskeletal modelling. The ‘goodness-of-fit’ of this approach was evaluated by comparing the resulting torques with the conventionally obtained experimental ones. To the best of our knowledge, this is the first study to investigate joint moment prediction via (neuro-)musculoskeletal modelling with statistical GRF and COP estimates. The secondary goal here is then to examine the individual effect of GRF and COP estimation accuracy on the joint moments predicted by each of the two models. The main contribution of this study lies in exploring the paradigm that enhances the capabilities of translating personalised model-based joint torque quantification of this type from laboratories to daily-life environments.The study has been validated on two male and three female healthy subjects  from a public dataset recorded by Schreiber and Moissenet . This daMarker trajectories and GRFM were passed to zero-lag second-order Butterworth low pass filters with cutoff frequency of 8 Hz . Marker In order to obtain individual joint torques, modelling analysis was performed using OpenSim —an open-In contrast to inverse dynamics which only considers musculoskeletal aspects, the EMG-assisted musculoskeletal model employed in this study consists of a neural-driven forward dynamics model and static optimisation element . This merameters  were assAs in , α was sribed in   to estimate the three-dimensional (3D) GRF and COP in anterio-posterior (AP) and medio-lateral (ML) direction. Since it is known that the performance of ANN models can be strongly influenced by the input suitability , a subseNCA is a nonparametric dimensionality reduction technique which learns the Mahalanobis distance used in the k-nearest neighbourhood classification algorithm . NCA hasAnalogous to the one-nearest-neighbour algorithm, NCA chooses point The average value of loss function is defined in Equation (4), in which he model .(4)li=∑For estimating each of the parameters described in Features selected by NCA were fed into separate ANN feedforward models for GRF and COP estimation in each direction. All the models were trained with one hidden layer ,55. The GRF was normalised to body weight (BW) and COP was analysed in millimetres. The ‘goodness-of-fit’ of GRF and COP estimates were assessed using the coefficient of determination To investigate the overall and individual effects of estimated GRF and COP on both non-neural and neural based sagittal torque prediction, three experimental conditions for each type of model were established. As such, the following combinations of inputs were fed into both musculoskeletal and EMG-assessed models: (i) both estimated GRF and estimated COP (EGEC-M/EGEC-N), (ii) estimated GRF and measured COP, (EGMC-M/EGMC-N) and (iii) measured GRF and estimated COP (MGEC-M/MGEC-N). Abbreviations of conditions with suffix ‘M’ and ‘N’ denote musculoskeletal and neuromusculoskeletal models, respectively. All the musculotendon and model parameters were tuned with measured data for each subject. The same set of optimised parameters and EMG recordings were used for all the experimental conditions. The concept of running the entire investigating paradigm for each subject is illustrated in the schematic flowchart . The torp = 0.05. All analyses were carried out with IBM SPSS .Shapiro–Wilk, Levene’s and Mauchly’s tests were conducted to examine the data normality, variance homogeneity and sphericity, respectively. Whenever these assumptions were met, a three-way mixed ANOVA was applied to determine whether there was significant difference in both torque metrics for factors: subjects, experimental conditions and models. In case of nonexistence of significant interaction effects among the factors, only main effects were analysed. If significant interaction is present, focused repeated measures ANOVA was conducted for each level of the factor(s) concerned to determine the simple main effects. In addition, another repeated measures ANOVA was conducted with subject means to test on the same hypotheses. In case significant difference was detected, post hoc paired t-tests with Bonferroni correction were performed for pairwise comparison. If the test assumptions were violated, nonparametric within-subject and between-subject Friedman tests were conducted, followed by post hoc Dunn–Bonferroni tests. Significance level was set at The average GRF and COP estimates from the ANN models across all the trials and subjects are depicted in The average joint torques computed via non-neural and neural models for all three experimental conditions, that is, (i) EGEC-M/EGEC-N, (ii) EGMC-M/EGMC-N and (iii) MGEC-M/MGEC-N, are illustrated in p < 0.046). The ground reaction estimates affected the two models to a similar extent at the knee joint for EGEC and EGMC. However, there was significant difference at the knee joint for MGEC on three subjects (p = 0.046). For ankle joint, significant difference was observed for EGEC and MGEC on all the subjects, and for EGMC on four subjects (p = 0.046). Statistical results from In the between-model comparison, RMSE results indicated that only a few significant differences were detected for hip joint for each of the conditions (0.025 < p < 0.04). At knee joint, significant difference was observed between EGEC-M and MGEC-M, and between EGEC-N and MGEC-N for Subject V (p = 0.04), while between EGEC-M and EGMC-M for Subject I (p = 0.04). However, subject means revealed no significant difference for all the conditions. At ankle joint, significant difference was detected between EGEC-M and EGMC-M, and EGEC-N and EGMC-N for Subject III and IV as well as mean values for both measures (0.005 < p < 0.04). Furthermore, Subject II showed significant difference in both metrics between EGMC-N and MGEC-N (p = 0.04). Generally, MGEC-M and MGEC-N performed better than EGMC-M and EGMC-N at hip joint; however, they did contrariwise at the ankle.In the between-condition comparison, The primary focus of this study was to investigate the feasibility of predicting joint torques in sagittal plane via both musculoskeletal and neuromusculoskeletal models with reduced reliance on ground reaction measurements (force platforms). The motivation behind this effort comes from a desire to allow subject-specific high-fidelity model-based gait analysis to take place outside the laboratory. We applied machine learning algorithms to estimate GRF and COP, which were then used to compute the torques via inverse dynamics and hybrid neuromusculoskeletal modelling. Moreover, the study considered the individual importance of GRF and COP estimation accuracy for torque prediction. To this end, we introduced two additional experimental conditions: feeding models with either (i) estimated GRF and measured COP or (ii) measured GRF and estimated COP. We evaluated the accuracy of all three experimental conditions for both models with respect to the corresponding measured values, and the proportion of measured torque variance that was explained by the ground reaction estimates.In the first part of this study, we estimated intrasubject 3D GRF as well as AP and ML COP using separate ANN models. The application of GRF/COP estimation using machine learning has been attempted by a number of studies ,37,38,39Our average rections ,37,39. Drections  estimaterections  where thrections . This coFor the primary focus of this study, joint torques were predicted via both inverse dynamics and the application of the hybrid model, using the obtained GRF and/or COP estimates. Given the condition in which both GRF and COP were estimated (EGEC), the RMSE results exhibited significant differences between the two models at both knee and ankle for at least two subjects. Notwithstanding the close tracking of the hybrid model to inverse dynamics , the grLee and Park  estimatei et al.  estimatei et al.  predicteIn addition to these investigations, the individual effects of GRF and COP estimates on the joint torque prediction via the two models were considered. To this end, four additional conditions (two for each model): EGMC-M, EGMC-N, MGEC-M and MGEC-N were investigated. For hip, statistical results in two of the five subjects showed that there was significant difference between EGEC and MGEC, suggesting that the inaccuracies from the GRF affected torque estimates more than those from COP. This, as a trend, was also evident in the average results. Contrariwise, the ankle torques were more sensitive to inaccuracies of COP than those of GRF, as observed from two of the five individuals, as well as from the average outcomes. For knee, it could not be concluded whether GRF or COP inaccuracies had a larger effect on joint torques, as only one significant difference was found in each of the cases. Moreover, an obvious positive relationship was observed between o ML GRF . Hence, Due to the exploratory nature of this study, we considered only five subjects, which limits investigations beyond those discussed here. Thus, a follow-up work on a larger subject pool would be valuable at this point. Another point to note is that this study used EMG recordings from eight muscles to run the neuromusculoskeletal model. While this corresponds to the setup of previous work , an inveThis study has investigated the feasibility of sagittal joint torque prediction based on both non-neural and neural musculoskeletal hybrid models with statistical ground reaction estimates. This approach enables consideration of subject-specific anatomy and neurophysiology via hybrid modelling with limited use of force plates, which favours gait analysis in laboratory-free settings. The applied method results in the least torque estimation error in ankle, and the most in hip for both models. Furthermore, the individual impact of the GRF/COP estimates on joint torques has been examined. The outcome suggests future focus on improving different ground reaction estimates, which depends on the joint(s) of interest. This study helps pave the way towards gait analysis of this type in daily-life activities.


In multivariate regression analysis, first-day NP was independently associated with p-CFL occurrence [odds ratio (OR)=6.395, 95% confidence interval (CI)=2.236-18.290, p=0.001). After adding the NP change into the regression model, first-day NP was no longer independently associated with p-CFL, and NP change  was independently associated with p-CFL. The receiver operating characteristic curve comparison analysis showed that NP change had a significantly better predicting value than first-day NP . NP is an effective imaging marker for predicting p-CFL after endoscopic sellar and suprasellar tumors operation, and the NP change has a better predicting value.We aimed to study the relationship between pneumocephalus on non-contrast CT (NCCT) and post-operative cerebrospinal fluid leakage (p-CFL) after endoscopic transsphenoidal sellar and suprasellar tumor surgeries. Data from patients who underwent endoscopic treatment for sellar or suprasellar tumors from January 2018 to March 2020 were consecutively collected and reviewed. The NCCT pneumocephalus (NP) was measured the first day after operation and the first day after the expansive sponge was extracted. p-CFL was determined according to post-operative clinical symptoms, high resolution CT and glucose test, and expert consensus. Of the 253 patients enrolled in this study, 32 (12.6%) had p-CFL. Compared with patients without p-CFL, patients with p-CFL had a higher occurrence of intra-operative CFL, a longer operation time, a higher rate of pneumocephalus on first-day NCCT after operation , and a higher rate of NP volume change between two NCCT measurements (referred to as the NP change) (all  Endoscopic transsphenoidal surgery is increasingly performed by neurosurgeons to treat skull base lesions, but cerebrospinal fluid (CSF) leakage (CFL) is a difficult-to-avoid complication, with an incidence as high as 11% . In one Early detection of CFL is quite important. Methods reported to diagnose CFL include β2‐transferrin testing, glucose rhinorrhea content analysis, high-resolution computed tomography (HRCT), magnetic resonance imaging (MRI), and cisternography –13. HoweHere, we analyzed surgical cases of endoscopic transsphenoidal sellar and suprasellar tumors, focusing on the relationship between CFL and the occurrence and volume change of NCCT pneumocephalus (NP) to identify a new method of CFL diagnosis.Each subject or an appropriate family member provided written informed consent prior to the study, and the protocols were approved by the local ethics committee. All clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki.The retrospective study included consecutively collected patients who underwent endoscopic treatment for sellar or suprasellar tumors from January 2018 to March 2020. Patients’ demographics, medical histories, pathological findings, and repair outcomes were recorded. Pre- and post-operative imaging, operative reports, medical records, and operative videos were reviewed. We excluded cases if (i) post-operative pathology did not show evidence of a tumor or (ii) they were missing imaging and clinical data Figure 1via lumbar cistern drainage; artificial dura repair; balloon compression; or autologous tissue like fat tamponade, fascia lata, or a pediculate nasoseptal flap . The NP volume measurement method is shown in The presence of NCCT pneumocephalus (NP) was measured on the first day after operation, and NP volume was estimated the first day after expansive sponge extraction. NP change was defined as the volume change in NP between the first day after operation and 24 h after sponge extraction, and it was divided into four categories: no NP in both NCCT measurements, NP volume decreased, NP volume remained and NP volume increased, which were abbreviated as no NP, decreased NP, unchanged NP and increased NP, respectively. The latter three categories were ascribed as NP change. NP delineation and volumetric analysis were conducted using MRIcron software (The diagnostic flow chart is shown in Post-operative CFL (p-CFL) was diagnosed if patients had➀Persistent post-operative nasal discharge regardless of the occurrence of intra-operative CFL;➁Neither intra-operative CFL nor persistent post-operative nasal discharge but positive HRCT finding (CSF leak through a defect);➂Non-persistent nasal discharge after surgery, negative HRCT, but positive glucose test (glucose rhinorrhea content ≥1.7 mmol/L). In that case, glucose was repetitively tested, and the p-CFL diagnosis was determined by two experienced neurosurgeons . When there were different diagnostic opinions, the neurosurgeons reached consensus after discussion.Patients without post-operative nasal discharge and negative CSF tests were defined as non-p-CFL.The characteristics of patients with and without p-CFL  are presented as mean and standard deviation (SD) for normally distributed continuous data, non-normally distributed variables are described as quartiles, and categorical variables are given as counts and percentages. Between-group differences were evaluated using Student’s t-tests or Mann–Whitney U-tests for continuous data and chi-square tests or Fisher’s exact tests for categorical variables.p < 0.05) were included in the binary logistic regression model. Adjusted odds ratios (ORs) with 95% confidence intervals (CIs) were calculated, and p < 0.05 was considered significant. Receiver operative curve (ROC) analysis was performed to test the predictive power of independent variables for the dependent variable; we calculated the areas under the ROC curve (AUC), sensitivity, and specificity. All analyses were performed after blinding the participants’ identifying information. Statistical analysis was performed using SPSS 19 . ROC curve comparison analyses were conducted by MedCalc statistical software version 15 .Variables that were significant on univariate analysis  post-operative pathology did not show evidence of a tumor (n = 12), and (ii) incomplete imaging and clinical data from admission to discharge (n = 6). Of the 253 patients, 49.6% were female, and the median patient age was 50 years .Of all the 253 patients, 40 (15.8%) showed first-day NP after operation. After the extraction of the expansive sponge, 46 patients (18.2%) showed NP change, and 32 (12.6%) were finally confirmed with p-CFL. The median test time for p-CFL was 2 days  after the removal of the expansive sponge, and 62.5% of p-CFL patients were tested beyond 2 days after the removal of the expansive sponge.2 = 48.090, p < 0.001) . After resecting tumors, patients with p-CFL showed a higher rate of first-day NP and NP change during hospitalization, a higher occurrence of high fever and infection, and a longer duration of hospitalization  , none was found of p-CFL in both NCCT measurements. Among patients with an NP change (n = 46), 10 were in the decreased-NP subgroup who initially showed first-day NP but not on follow-up NCCT and were determined not to have p-CFL. Sixteen patients who showed increased NP between both NCCT measurements were confirmed to have p-CFL, including six patients without NP on first-day NCCT but with NP on follow-up NCCT after sponge extraction Figure 1When adding first-day NP into the multivariate regression model, both the first-day NP  and operative duration time  were independently associated with the occurrence of p-CFL after adjusting for pathology type  and operative duration time  were independently associated with p-CFL , with sensitivity of 68.6% and specificity of 87.8%. The AUC of NP change for predicting p-CFL was 0.988 , with sensitivity of 100% and specificity of 93.7%. The ROC comparison showed that NP change had a significantly better predicting value than first-day NP .Klebsiella pneumoniae infection and died. Thus, it is vital to find early signs that can be used to diagnose CFL. We found that both first-day post-operative NP and its volume change over time could predict p-CFL, and the latter had a higher predictive value. Compared with patients who had no NP, those with no change and increased NP had a higher risk of having p-CFL, and 100% of increased-NP patients (n = 16) were confirmed to have p-CFL.Diagnosis and management of p-CFL can be challenging, even for the most experienced neurosurgeons . Clear rThere are two possible pathophysiologic explanations for the correlation between the presence of NP and p-CFL: the inverted bottle mechanism and the ball-valve mechanism , 27, 28.We found that the NP change after expansive sponge extraction could better predict p-CFL than the first-day NP. Previous studies considered post-operative NP as an epiphenomenon of intra-operative CSF leak which would correlate with a higher risk of p-CFL , 32. HeaIn patients with first-day NP after tumor resection, the occurrence of p-CFL was lower among those with reduced NP volume compared with those with increased or unchanged NP volume. The probable cause is that pneumocephalus was gradually absorbed after the CFL resolved. Intra-operative CFL and the loss of a large amount of CSF initially leads to pneumocephalus. However, when the CFL was properly repaired with skull base reconstruction, the post-operative pneumocephalus gradually absorbed. In contrast, no change or an increase in CT pneumocephalus suggested persistent CFL. If the CFL is completely solved, the pneumocephalus will be gradually absorbed, and CT re-examination after 3 days is sufficient to observe the pneumocephalus reduction. If there is no change or there is an increase in pneumocephalus volume post-operatively, it indicates that intracranial pressure continues to decrease and a leak still exists, so the incidence of p-CFL increases.via lumbar cistern drainage.In this study, p-CFL developed in two patients who had no pneumocephalus on the first day after the operation. Pneumocephalus was found on imaging on the fourth day after the operation, suggesting that dynamic review of head NCCT has supplementary significance for CFL evaluation. Although there was an increase in pneumocephalus volume after operation in one case, there was no CSF leakage, possibly due to excessive drainage of CSF Our study was limited by the small number of patients and single-center retrospective design, so multicenter studies with larger samples sizes are needed to confirm our findings. In addition, we did not use beta-2 transferrin or beta trace protein testing to diagnose CFL because our hospital does not routinely perform these analyses. Nevertheless, glucose rhinorrhea content testing is fast, simple, inexpensive, and can be carried out in most hospitals, which could promote the wide application of our findings. An evidence-based review also pointed out that the guidance level for glucose testing was not inferior to that of beta-2 transferrin or beta trace protein testing in identifying CFL . FinallyOur results show that NP change is a more convenient and effective imaging marker than first-day NP for predicting p-CFL after endoscopic sellar and suprasellar tumor resection. During dynamic NCCT observation, attention should be paid to the risk of p-CFL in patients with no change or increased NP.The original contributions presented in the study are included in the article/This study was approved by the ethics committee of The second Affiliated Hospital of Zhejiang University, School of Medicine. The patients/participants provided their written informed consent to participate in this study.WG, XW and SZ: drafted the manuscript. YF: acquisition of data. SZ: analysis or interpretation of data. CL, YH and WY: study concept and design. SZ and CL: help to revise the whole framework and polish the language.This work was supported by the National Natural Science Foundation of China (grant number 81801162), the Zhejiang Provincial Natural Science Foundation of China (grant number LGF22H090020) and the Medical Health Science and Technology Project of Zhejiang Provincial Health Commission (grant number 2022KY600).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


There have been several calls for estimations of costs and consequences of headache interventions to inform European public-health policies. In a previous paper, in the absence of universally accepted methodology, we developed headache-type-specific analytical models to be applied to implementation of structured headache services in Europe as the health-care solution to headache. Here we apply this methodology and present the findings.Data sources were published evidence and expert opinions, including those from an earlier economic evaluation framework using the WHO-CHOICE model. We used three headache-type-specific analytical models, for migraine, tension-type-headache (TTH) and medication-overuse-headache (MOH). We considered three European Region case studies, from Luxembourg, Russia and Spain to include a range of health-care systems, comparing current  care versus target care . We made annual and 5-year cost estimates from health-care provider and societal perspectives . We expressed effectiveness as healthy life years (HLYs) gained, and cost-effectiveness as incremental cost-effectiveness-ratios . We applied WHO thresholds for cost-effectiveness.The models demonstrated increased effectiveness, and cost-effectiveness (migraine) or cost saving  from the provider perspective over one and 5 years and consistently across the health-care systems and settings. From the societal perspective, we found structured headache services would be economically successful, not only delivering increased effectiveness but also cost saving across headache types and over time. The predicted magnitude of cost saving correlated positively with country wage levels. Lost productivity had a major impact on these estimates, but sensitivity analyses showed the intervention remained cost-effective across all models when we assumed that remedying disability would recover only 20% of lost productivity.This is the first study to propose a health-care solution for headache, in the form of structured headache services, and evaluate it economically in multiple settings. Despite numerous challenges, we demonstrated that economic evaluation of headache services, in terms of outcomes and costs, is feasible as well as necessary. Furthermore, it is strongly supportive of the proposed intervention, while its framework is general enough to be easily adapted and implemented across Europe.The online version contains supplementary material available at 10.1186/s10194-021-01305-8. Despiteg, , these deg, . The soleg, , 4, in aeg, . In a laeg, . Here weIndirect costs are a key issue in economic evaluation. Because headache disorders are disabling –9, lost The methods are described in detail in the earlier paper . We modeOutcomes were measured in healthy life years (HLYs), and effectiveness as HLYs gained by change from current to target care. We assumed that target care would partially but not entirely close treatment gaps : provideCosts included health-care costs  from the provider perspective; additionally, lost productivity (days lost from work) was included in estimates made from the societal perspective. Methodological details are provided elsewhere on the decision-analytical models, on epidemiological data (including disability), estimations of intervention effectiveness, economic outcomes (including use of resources and lost productivity), treatment management plans and selection of interventions for migraine, TTH and MOH within the alternatives under comparison .Economic and effectiveness outcomes were brought together to evaluate cost-effectiveness in terms of costs to be invested per HLY gained , with the three health-care systems of Russia, Luxemburg and Spain bringing different systems of health-care service delivery and financing into the model.Limited evidence supports opportunity-cost–based cost-effectiveness thresholds applicable across diverse countries, including those of interest here. We applied WHO’s thresholds against gross domestic product (GDP) for this purpose: interventions costing < 3*GDP per capita per HLY gained were cost-effective, those costing < 1*GDP per capita per HLY gained were highly cost-effective . AlthougThe principal analyses were conducted from the health-care provider perspective, with robustness tested in a series of sensitivity analyses inflating health-care costs and deflating HLYs gains while keeping to the same cost-effectiveness thresholds. In a series of secondary analyses, we considered the larger societal perspective. For the baseline societal analysis, we assumed all lost productivity was explained by disease-attributed disability, whereas, in a conservative alternative, we assumed that this disability accounted for only 20% of lost productivity (so that only this proportion might be recovered).Summaries of the economic and effectiveness outcomes for the treatments of each headache type are presented in Tables In short-term modelling (1-year time frame) from the health-care provider perspective, the intervention was found to be cost-effective for migraine Fig.  – well bFrom the health-care provider perspective, the hypothetical shift to target care would bring gains in HLYs (the longer the time frame the greater the gain). For migraine, resources must be invested to secure these benefits . For TTH and MOH, the benefits would be accompanied by cost savings (the longer the time frame the greater the economic gain). Findings were consistent across the health-care systems of the three countries.From the societal perspective .Finally, we considered cost and effectiveness outcomes for 1000 patients with migraine, 1000 with TTH and 1000 with MOH patients in each country setting, allowing comparisons of the impact of introducing target care across country-specific populations . For TTOf course, these findings assumed that lost productivity reported as a consequence of headache would, therefore, be recovered commensurately as headache was alleviated. There was reason to doubt this assumption, since many extraneous factors influence the relationship between headache-attributed disability and lost productivity , 15. It There have been repeated calls for better modelling of costs and outcomes of headache interventions to inform public-health policies, given the very high prevalence of headache disorders , 21–23. The principal limitations of this study were those inherent in economic modelling. We were dependent on the type and quality of the data sourced in order to calculate the economic outcomes, the latter being imperfect in Eurolight . SimilarEven with very conservative assumptions, highly inflating costings (or deflating expected gains), we could conclude that structured headache services would be cost-effective according to WHO thresholds  – and thThe framework of the proposed intervention is general enough to be easily adapted and implemented . Thus, sStructured headache services – offering care efficiently and equitably to the widest number of people  and, accAdditional file 1: Appendix 1. Baseline vs sensitivity analyses: Luxembourg. Appendix 2. Baseline vs sensitivity analyses: Russia. Appendix 3. Baseline vs sensitivity analyses: Spain. Appendix 4. Luxembourg: economic results of changing from current to target care (population estimates). Appendix 5. Russia: economic results of changing from current to target care (population estimates). Appendix 6. Spain: economic results of changing from current to target care (population estimates). Appendix 7. Differences in outcomes when changing from current to target care (cohorts of 1000 patients per type of headache in Luxembourg). Appendix 8. Differences in outcomes when changing from current to target care (cohorts of 1000 patients per type of headache in Russia). Appendix 9. Differences in outcomes when changing from current to target care (cohorts of 1000 patients per type of headache in Spain).


The unprecedented COVID-19 pandemic has led to lockdowns across the world with people being separated from their loved ones including partners, family, and friends. Here, using a large sample of 1,749 Australians and Americans, we investigated the impact of COVID-19 isolation on younger populations (13–25 years), and the influence of coping strategies and mental well-being on this impact. Overall, COVID-19 isolation had a more negative impact on adolescence (13–17 years) than young adulthood (18–25 years), but with no difference apparent between men and women, or between Australian and American residents. However, a deeper analysis revealed a gender-specific effect: the type of coping strategies differentially influenced the negative impact of COVID-19 isolation on men with various levels of well-being, an interaction effect not apparent in women. For men with lower levels of mental well-being, COVID-19 isolation appeared to have a less negative impact on them if they used more approach-oriented coping strategies . Our results provide cross-sectional evidence for a differential impact on young men at low levels of wellbeing by pandemic isolation. In sum, young men and adolescent boys with lower well-being coped better with COVID-19 isolation when they used more approach coping strategies. The COVID-19 pandemic has drastically changed our lives. People across the world have been isolated for months or more, and physical contact with individuals outside of their homes has typically been discouraged or prohibited. Isolation often means separation from loved ones including family, friends, or partners, which can result in an increase in distress in any population , and exaA major factor that may contribute to the extent to which one is negatively affected by isolation is the ability to maintain optimal well-being. Well-being can be defined as a combination of both subjective and psychological well-being; for instance, happiness, life satisfaction, positive relations with others, and goal-directed behavior . ResearcAnother potential predictor for the level of distress during the pandemic is the type of coping strategies used under stressful situations. Previous studies have shown that people who rely on avoidance-oriented coping strategies  have a heightened probability of exhibiting psychiatric symptoms compared to people relying on active coping styles such as active problem solving , 22. ConIn the current study, we will explore how well-being and the type of coping strategies interact in influencing the impact of COVID-19 isolation on a sample of 1,749 community participants aged from 13 to 25 years across Australia and the USA. Here we focus on the avoidance-oriented and approach-oriented coping styles. We expect that individuals with higher well-being should be more resilient to the negative impact of isolation than people with lower well-being. In addition, because avoidance behavior has been linked with increased psychiatric symptoms after isolation, whereas maintaining social connectedness is associated with reduced negative feelings, we predict that adopting a more active coping style would help reduce the negative impact of isolation. We therefore predict that approach coping strategies would benefit individuals who demonstrate lower levels of well-being in particular, in terms of coping with the effect of separation. Finally, since previous findings are mixed regarding whether demographics is predictive of the psychological outcomes of isolation , 32, we via Qualtrics survey panels of community participants residing in Australia or the USA. The Qualtrics Team sent database members of online Qualtrics survey on our behalf to selected members. Participant recruitment focused on three defining criteria: age , gender , and country of residence . Inclusion criteria included English as a primary language. Parental/guardian consent for 13–17 year old participants was first obtained using the opt-out approach, followed by implied consent of the participant when they completed the survey. Participants were asked to complete a 20-min survey on four occasions (every 6 weeks) over a 6-month period, although they were able to opt out of the study at any time. For Qualtrics panels, all of the participants are assured confidentiality, and they are anonymous to the researchers. To exclude duplication and ensure validity, Qualtrics checks every IP address and uses a sophisticated digital fingerprint technology. In addition, every strategic panel partner uses deduplication technology to provide the most reliable results and retain the integrity of the survey data. They adopt Relevant ID in addition to this which has the unique ability to identify multiple panel accounts from different research firms on the same computer. Suspect respondents are flagged in the system and are either allowed, redirected or completely filtered out of surveys in which they attempt to participate. The Qualtrics Team reimbursed participants for their participation by means of a small incentive  per survey completion. The study was approved by the Human Research Ethics Committee of the University of New South Wales (UNSW), Sydney, Australia (HC200150).Survey data was collected in a sample of 2,580 younger participants (13–25 years) n = 831) who provided positive-impact ratings for events that should have been rated as negative . The final sample included 1,749 participants ranging in age from 13 to 25 years , 656 men/1,093 women, of which 944 participants resided in Australia and 805 in the US  Temporary separation from girlfriend/boyfriend due to COVID-19?; (2) Temporary separation from your close friends due to COVID-19?; and (3) Temporary separation from your immediate family due to COVID-19?”. If the answer was yes to any of these questions, the participants then rated the positive vs. negative impact this experience had on their life using a 5-point scale . Mean scores were used to quantify the impact of isolation.Predictors: COMPAS-W scores, Brief-COPE scores (Avoidance/Approach), age, gender, country, and the interactions of these factors; Outcome: COVID-19 isolation impact ratings). In the statistical analysis, age, gender, and country of residence were included as predictors, and we also controlled for the effect of marital status, employment, family income, and history of psychological disorders in all models. In our analyses, we treated age as a categorical variable with two levels based on developmental periods: adolescence (13–17 years) and young adulthood (18–25 years). Linear mixed-models were used to explore any significant effects found for the predictor variables. Multiple comparisons were Bonferroni-corrected for significant interaction effects (p < 0.008). All analyses were two-tailed. Statistics were performed using MATLAB R2020a.To examine how younger people's well-being, coping strategies and demographics affect their experience during the COVID-19 isolation, we tested whether the variance explained by these variables were significantly greater than the unexplained variance using omnibus regression models (t-tests showed that (1) adolescents scored significantly higher on COMPAS-W than young adults ; (2) male participants scored significantly higher on COMPAS-W than female participants ; with (3) no significant difference between Australians and Americans  for well-being.We first examined the level of well-being during the COVID-19 pandemic for our sample as a function of age, gender, and country of residence. The mean COMPAS-W well-being scores were 96.18 (SD = 13.95) for adolescents (13–17 years) and 88.48 (SD = 14.28) for young adults (18–25 years); 91.85 (SD = 14.45) for women and 95.33 (SD = 14.51) for men; and 92.49 (SD = 13.92) for Australians and 93.93 (SD = 15.27) for Americans. Using a Bonferroni-corrected significance threshold of 0.017 (0.05/3), two-sample r = −0.35; p < 0.001], and a significant positive correlation between COMPAS-W well-being scores and Approach coping scores . This suggests individuals who use more approach coping strategies tend to have higher well-being, whereas those who use more avoidance coping strategies tend to have lower well-being.Next we checked the relationship between well-being and coping strategies. According to the findings of previous studies, the tendency of using Avoidance coping is associated with negative psychological outcomes, whereas the tendency of using Approach coping is associated with positive psychological outcomes . Consistt = −14.24, p < 0.001]. Adolescents also used significantly less Approach coping than young adults . Women  used significantly more Avoidance coping than men , and their use of Approach coping did not differ significantly . The use of Avoidance coping did not differ significantly between Australians  and Americans  = −1.81, p = 0.070). Americans  used significantly more Approach coping than Australians .Whether the coping strategies individuals used differ by their age, gender, and country of residence was then examined. Using a Bonferroni-corrected significance level of 0.017 (0.05/3), adolescents  used significantly less Avoidance coping than young adults , suggesting that COVID-19 isolation indeed had a negative impact on younger people's lives overall.We next examined the general impact of COVID-19 isolation (using an average of responses to the three COVID-19 separation questions) relative to the baseline of zero (indicating “no impact”) on our sample. A one-sample t = −3.54, p < 0.001]. Furthermore, although men  and women  rated COVID-19 isolation as a negative experience, the ratings did not differ significantly between them . Finally, both Australians  and Americans  rated COVID-19 isolation as having a negative impact, but again the impact did not differ significantly between the two countries . These results suggest that COVID-19 isolation has a negative impact on individuals regardless of their demographics, and that it appears to more negatively affect adolescents than young adults.To check whether COVID-19 isolation had a differential impact on different demographic groups, we separately analyzed age, gender, and country of residence. Using a Bonferroni-corrected significance threshold of 0.017 (0.05/3), both age groups rated COVID-19 isolation as being a negative experience , but the impact was significantly more negative for adolescents [p = 0.622). Similarly, there was no significant interaction of COMPAS-W well-being scores, Approach coping, age, gender, and country of residence . The interaction of COMPAS-W well-being scores, coping and country of residence was also not significant . The interaction of COMPAS-W well-being scores, Avoidance coping and gender was again not significant , but that of COMPAS-W well-being scores, Approach coping and gender was marginally significant  contributed to the influence of well-being and approach coping strategies on the impact of COVID-19 isolation . The BonPost-hoc comparisons revealed that COVID-19 isolation impact ratings were significantly lower in the LCLW group compared to HCLW groups . By contrast, there was no significant difference in isolation ratings for LCHW and HCHW groups . These results suggest that the pandemic seems to have a more negative impact on younger men who do not seem to have an effective coping strategy, but only for those with a lower level of well-being.To dissect the interaction of well-being and Approach coping, we used the medians of COMPAS-W and Brief-COPE (Approach) scores to classify the male sample into four groups: low-coping-low-well-being (LCLW), high-coping-low-well-being (HCLW), low-coping-high-well-being (LCHW), and high-coping-high-well-being (HCHW) groups. p = 0.001; p < 0.001; p = 0.004; p = 0.081; p = 0.403; p = 0.029; Since Approach coping is a broad strategy containing six specific subscales, we further examined whether specific approach coping strategies were more effective in alleviating the negative impact of isolation on young men than others. Using the Bonferroni-corrected significance level of 0.008 (0.05/6), we found significant interactions of COMPAS-W scores and scores for active coping , positive reframing , and planning , but they did not significantly differ between LCHW and HCHW groups . These results suggest that active coping, positive reframing and planning are effective in reducing the negative impact of COVID-19 separation on men with low levels of mental well-being.The present study investigated the influence of mental well-being and coping strategies on how COVID-19 isolation affects younger populations. Using a large sample of 1,749 younger Australians and Americans (13–25 years), we found a gender-specific effect on the isolation experience during the pandemic associated with the type of coping strategies and well-being level. Specifically, approach-oriented coping strategies, especially active coping, positive reframing, and planning, appeared to mitigate the negative influence of COVID-19 isolation on young men at low levels of wellbeing. By contrast, there was no evidence of coping strategies having differential effects on the negative experience of COVID-19 isolation for young women. Together, our study suggests that an unexpected stressor such as a pandemic can differentially affect young men and women, especially young men who have low well-being but can actively cope with the stress.Approach-oriented coping strategies seem to only benefit a specific subset of our sample: young men with lower well-being. By contrast, the effect of approach coping did not manifest in young men who reported having high well-being. This effect is likely the result of the fact that individuals with higher well-being inherently adopt more adaptive coping strategies such as approach coping by default, which does not change regardless of the situation or levels of stress. This is evident by the significant correlations we reported between well-being and different coping strategies. Therefore, individuals who scored high on well-being were likely to use more effective coping strategies, and the benefit may have buffered them from the negative impact of isolation that was reported by others –50. On tOur results show that male and female participants reported similar negative ratings for COVID-19 isolation, suggesting a similar degree of negative impact of the isolation across men and women. Ostensibly, this contradicts with previous studies reporting that females showed increased distress during the COVID-19 pandemic [, 51, 52;Deeper analyses revealed that some specific approach coping strategies appeared more effective than others during COVID-19 isolation. Previous studies have shown the effectiveness of actively focusing on a problem in reducing both distress and the likelihood of exhibiting psychiatric symptoms , 54, 55.Our results show that adolescents are more negatively influenced by COVID-19 isolation compared to young adults. This aligns with previous findings showing that younger age is a risk factor under stressful circumstances , 52, 59,In addition to gender and age, another demographic factor we considered was country of residence. We did not find evidence of a differential impact of COVID-19 isolation on Australians vs. Americans. However, we note the cross-sectional nature of our study and the timing of it. Our baseline surveys were conducted in May and June when the COVID-19 had been plaguing the two nations for a similar amount of time. In contrast, it was later on during July when the rates of COVID-19 infection reduced significantly in Australia and restrictions in most states were lifted, whereas in the US infection rates have kept skyrocketing beyond the current study's baseline data collection period. Another possible interpretation for the similar ratings is the increased reliance on approach coping strategies by Americans compared to Australians, which is apparent in our results. This might have helped Americans cope with a more severe pandemic. But due to the continuously high infection rates in the US, we speculate that the current results will change to reflect this when we examine longitudinal change over time. Thus, it is important for future studies to examine cross-country differences over time.A seemingly counterintuitive finding of the current study is that some participants rated the experience of COVID-19 isolation as being positive. The interpretation is likely to be multifaceted. One factor might be a “fear of infection,” which includes both fears about one's own health and the possibility of infecting others , 66–69. via multiple channels such as education and media propaganda, in order to elevate overall well-being in the general population. When future pandemics or adversities arise, policy makers should consider the differential impact of well-being and coping mechanisms on men and women, and prioritize interventions and resources for vulnerable groups, for example, young men, adolescents, and individuals with low levels of well-being.The present study has implications for both clinicians and policy makers. In clinical settings, it is imperative to individualize behavioral therapies and treatments, especially for young men, in accordance with their levels of well-being. This may potentially increase the effectiveness of the treatments as young men seem to respond differentially to active coping mechanisms depending on their ability to maintain a healthy level of well-being under stressful circumstances. This health strategy could be accompanied by policies to emphasize the importance of well-being over and above illness symptoms alone; that is, the importance of living a healthy and balanced life There are three caveats here. First, the current study focused on adolescents and young adults. Future studies could use older populations and examine whether there is a gender-specific well-being effect on coping effectiveness. Second, the present study is cross-sectional. It would be crucial to systematically examine the longitudinal impact of COVID-19 isolation on mental well-being. A longitudinal design also allows investigating the effectiveness/quality of certain coping strategies over time for promoting well-being. Finally, our study did not include the duration of separation from loved ones when measuring the impact of COVID-19 separation. It would be interesting to examine how the separation duration affects the psychological consequence of COVID-19 lockdown.In conclusion, using a large sample of 1,749 Australians and Americans, the present study provides cross-sectional evidence that approach-oriented coping strategies can mitigate negative impacts of COVID-19 isolation on young men with low well-being. Our results indicate that approach coping is particularly useful in mitigating isolation distress in young men with lower levels of wellbeing. Our study calls for future research to compare the impact of COVID-19 isolation between men and women over time, and to identity the gender-specific and gender-general predictors for differential outcomes.j.gatt@neura.edu.au.The datasets presented in this article are not readily available due to ethical requirements. Requests to access the datasets should be directed to Justine M. Gatt, This study was reviewed and approved by University of New South Wales HREC (Project number: HC200150). Written informed consent to participate was provided either by the participants if 18 years of age or above, or by the participants' parent/legal guardian if under the age of 18.PC and HP analyzed the data. PC drafted the manuscript. HP and JG assisted with interpretations of the study results and provided revisions to all drafts. JG coordinated the overall project and supervised the research program. All authors contributed to the conceptualization of the current study's aims.JG holds stock in Map Biotech Pty Ltd. The remaining 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.