To quantify inequalities in zoster vaccine uptake by determining its association with socio-demographic factors: age, gender, ethnicity, immigration status, deprivation (at Lower-layer Super Output Area-level), care home residence and living arrangements.This population-based cohort study utilised anonymised primary care electronic health records from England  linked to deprivation and hospitalisation data. Data from 35,333 individuals from 277 general practices in England and eligible for zoster vaccination during the two-year period (2013–2015) after vaccine introduction were analysed. Logistic regression was used to obtain adjusted odds ratios (aOR) for the association of socio-demographic factors with zoster vaccine uptake for adults aged 70 years (main target group) and adults aged 79 years (catch-up group).Amongst those eligible for vaccination, 52.4%  received the vaccine. Socio-demographic factors independently associated with lower zoster vaccine uptake in multivariable analyses were: being older (catch-up group: aged 79 years) aOR = 0.89 :0.85–0.93), care home residence (aOR = 0.64 (95%CI: 0.57–0.73)) and living alone (aOR = 0.85 (95%CI: 0.81–0.90)). Uptake decreased with increasing levels of deprivation ). Uptake was also lower amongst those of non-White ethnicities ) but was not lower among immigrants after adjusting for ethnicity. Lower uptake was also seen amongst females compared to men in the catch-up group.Inequalities in zoster vaccine uptake exist in England; with lower uptake among those of non-White ethnicities, and among those living alone, in a care home and in more deprived areas. Tailored interventions to increase uptake in these social groups should assist in realising the aim of mitigating vaccination inequalities. As care home residents are also at higher risk of zoster, improving the uptake of zoster vaccination in this group will also mitigate inequalities in zoster burden. Zoster is caused by reactivation of latent varicella-zoster virus infection and mainly affects older individuals. It is characterised by a painful dermatomal rash which may be followed by persisting pain called post-herpetic neuralgia (PHN). Amongst Monitoring and reducing inequalities in healthcare services or interventions is a statutory requirement in the UK. InequaliAscertainment of the socio-demographic determinants of zoster vaccine uptake can provide important information to public health professionals to address vaccination-related inequalities and reduce zoster disease burden. We have recently shown that routinely collected clinical and administrative information in the form of anonymised linked electronic health records are a useful resource to examine some of these socio-demographic factors, and these data can be used to supplement the routine surveillance data.The primary objective of this study was therefore to identify the socio-demographic determinants, of zoster vaccine uptake in England, using one of the world’s largest databases of general practice electronic health records: the Clinical Practice Research Datalink (CPRD), with an The CPRD provides quality-assured anonymised primary care patients’ clinical, administrative and lifestyle data representative of the UK population and covering approximately 7% of general practices from England., 17 Addist July) to assign study participants’ day and month of birth would wrongly classify some individuals as eligible for zoster vaccination. Importantly, the resulting unvaccinated group would comprise a mixture of individuals with possibly differing social factors: a) those eligible for vaccination who chose not to be vaccinated and b) those ineligible on the grounds of age, thus potentially resulting in biased effect estimates. To address this, we selected all individuals born in 1943 (or 1934 for catch-up cohort), who would have been eligible for vaccination at some point during the 2-year follow-up period as follows: those born in January-August 1943 would be eligible for the vaccine in 2013/14 or in 2014/15 if born September-December 1943; and determined vaccine uptake for the 2-year study period. The study population therefore comprised individuals born in 1943 (the routine cohort) and in 1934 (the catch-up cohort), who were alive and registered on 01/09/2013  with a CPRD general practice that had agreed to linkage to HES and IMD data and that met CPRD’s quality assurance criteria.[This 2-year cohort study from England spanned the period from 01/09/2013 to 31/08/2015, the first two years after the zoster vaccine was introduced. To maintain patient anonymity, CPRD data provide only year of birth for adult patients. This posed a problem in how to identify individuals who were eligible for zoster vaccination, which is determined by their age on a specific date. The common convention of using the mid-year  were encouraged to co-administer zoster with seasonal influenza vaccine (SIV). We thereWe also identified, throughout the study period, individuals who had immunosuppressive conditions or treatments that were contraindications to receipt of zoster vaccine. This was done to identify those who were eligible to receive the live zoster vaccine for the main analysis, and to describe the extent of inadvertent administration of zoster vaccine to those with contraindications. The immunosuppressive conditions and drugs included were those listed in the UK Green Book; code lists  and algoA past history of zoster was also ascertained using zoster or PHN codes from both CPRD and HES  occurrinA complete case analysis using multivariable logistic regression was used to determine the association of socio-demographic factors with zoster vaccine uptake, using adjusted odds ratios (aOR) with 95% confidence intervals (CI). Logistic regression models were chosen to address the problem of potential misclassification of individuals for vaccine eligibility based on their date of birth and therefore the lack of information on person-time at risk for vaccination. The exposure and outcome characteristics of individuals excluded from complete case primary analysis because of missing data were described.a priori confounders of the socio-demographic factors of interest, as well as potential determinants of zoster vaccine uptake. An existing conceptual framework[Gender and being a part of the routine or catch-up cohort  were considered to be framework was adapframework Using thframework, 7 We thIn sensitivity analyses, we repeated multivariable analyses: 1) restricted to individuals who were followed up for the entire study period of two years; 2) adding the social factors that had been excluded due to multicollinearity issues; 3) replacing the status of the time-varying factors to that ascertained at the end of follow-up instead of at the start of follow-up, and 4) including past history of zoster in the final multivariable model as a potential mediator of some of the socio-demographic factors.Additionally, inadvertent zoster vaccination amongst individuals with immunosuppressive conditions or therapies was also described.Data were analysed using Stata 14 .Approval was sought and obtained from the Observational/Interventions Research Ethics Committee of the London School of Hygiene and Tropical Medicine (Reference: 11910) and from the Independent Scientific Advisory Committee (ISAC) of the Medicines and Healthcare products Regulatory Agency (reference:16_168). The ISAC protocol was made available to the reviewers of this paper.A total of 39,120 individuals born in 1943 or 1934 and with no evidence of prior zoster vaccination were registered with a CPRD practice, which had consented to linkages, on 01/09/2013 . The perA slight majority of the participants in the primary analysis  were femOf the total participants considered for the primary analysis, zoster vaccine was administered to 18,499 (52.4%) individuals. Uptake amongst the main target group (those born in 1943) was 53.4% compared to 50.8% amongst the catch-up cohort . Nearly half  of the participants received both zoster vaccine and SIV ; of thesA decision was made to drop marital status from the multivariable analyses, due to a large proportion of individuals with missing data for this variable. Thus, in the subsequent complete case analysis, only patients with missing ethnicity data  were excluded. This resulted in a final study population of 31,449 (89%) individuals, amongst whom the zoster vaccine uptake was 53.5% . CompariTime-varying exposures at the start and end of follow-up remained largely unchanged for 98.8% individuals included in the complete case analysis .a priori confounders (gender and year of birth), there was strong evidence of an association between higher zoster vaccine uptake and male gender, with uptake 10% higher compared to females . There  females .After additionally adjusting the minimally adjusted model for immigration status and ethnicity  . The resSubstitution of cohabitation status instead of living alone, practice- LSOA-level IMD instead of patient-LSOA-level IMD, excluding individuals with immunosuppressive conditions at the end as opposed to start of follow-up  and deteTo assess the impact of excluding individuals from the complete case analysis, a further exploratory analysis was conducted, by re-running the minimally adjusted analysis for the entire study population  with follow-up ≥5 months including those with missing data on ethnicity . CompariOf the 19,330 in the total study population who received vaccination , 3% . A graded inverse association of patient-level deprivation with vaccine uptake was also observed. Lower uptake was also seen amongst females in the catch-up cohort, and the effects of care home residence and deprivation were more marked among the older catch-up group.Strengths of the study include the large sample size, and linkages to hospitalisation data which provided additional information about socio-demographic factors as well as zoster vaccine contraindications. For multivariable analysis, a hierarchical approach using conceptual framework, enabled appropriate interpretation of effect estimates. The NHS The study limitations include potential misclassification of both socio-demographic factors and the zoster vaccination recording. Our categorisation of individuals without a relevant codes for a binary socio-demographic variable as not having the characteristic could have introduced errors; if this misclassification was non-differential with respect to vaccine uptake, this may have underestimated the effect estimates in this study. However, our previous methodological study showed good capture of socio-demographic factors in CPRD data when compared to the 2011 English Census. SimilarlOther factors indicative of socio-economic status of an individual and associated with vaccine uptake amongst older individuals, as reported by our systematic review, such as It is also feasible that individuals who see their GPs more frequently may have greater opportunity to be offered vaccination. Thus, it is possible that healthcare utilisation may be a potential mediating variable for the association between socio-demographic factors such as deprivation, male gender (in the catch-up cohort) or ethnicity and zoster vaccine uptake. If this were the case, adjusting for this mediating variable could attenuate the association between some of the socio-demographic factors and vaccine uptake.Although we were able to examine whether there was evidence of clustering at practice-level, lack of information on practice-level variables (other than practice-LSOA-level-IMD) prevented further investigation of this. It is possible that variations in certain general practice-level characteristics, for example staffing levels, opening hours, and call/recall processes, could have affected implementation of the zoster vaccine programme for all zoster vaccine-eligible patients in that practice. These general practice-level data were unavailable in CPRD and therefore we could not determine to what extent the lower uptake we found in specific groups related to lack of opportunity to be vaccinated rather than individual choice not to come forward for vaccination. However, it is notable that practice-level clustering had little impact on most of the effect estimates studied other than some of the estimates for ethnicity. Future studies assessing the effects of these factors on vaccine uptake may provide a better understanding for the reasons of lower uptake amongst specific population groups.Our finding of higher uptake in the main target group compared to the catch-up cohort reflects the findings from the national annual zoster vaccine coverage data for England over the same time period, and vaccine coverage among the individuals in our study who were followed up for the entire study duration (and thus had fuller capture of uptake of vaccination) is comparable to the coverage estimates in the national data., 7 HigheSeveral of our findings are novel with respect to zoster vaccine uptake. These include lower uptake among individuals who were living alone or not cohabiting; this echoes studies showing lower uptake of seasonal influenza vaccine among those living alone in older European populations. Our findThis study demonstrates that in a public funded healthcare system, vaccination inequalities exist during a crucial period of programme initiation, and identifies socio-demographic groups that could be targeted with tailored interventions to increase zoster vaccine uptake. Of particular interest is the finding of lower uptake among care home residents; we have shown recently that individuals in care homes are at higher risk of developing zoster, and so aSimilarly, targeting of older individuals who live alone may be needed to encourage zoster vaccination. Individuals cohabiting or living with their relatives may be more motivated by their social networks to get vaccinated. Secondly, higher disease awareness amongst these individuals, by witnessing the debilitating effect of zoster in their relatives, may also increase uptake. This was examined in a US study that reported higher zoster vaccine uptake amongst individuals in the three months after occurrence of zoster in their partners, reflecting a short term effect of disease awareness. However,This population-based cohort study provides evidence of inequalities in zoster vaccine uptake in the period immediately after the introduction of a national vaccination programme, identifying a wide range of socio-demographic determinants of uptake of zoster vaccine. This work should encourage further research into the reasons why specific socio-demographic groups are less likely to receive zoster vaccine, and effective planning and implementation of specific interventions to target these socio-demographic groups to mitigate vaccination inequalities amongst older individuals. Factors that are currently poorly recorded in routinely collected data, such as religion, education and income, should inform policy drivers such as the sustainability and transformation partnerships to incentivise better recording of these factors and/or facilitate other data linkages for comprehensive knowledge and future interventions to improve overall health and wellbeing of older populations. As care home residents are both less likely to receive zoster vaccine and are at higher risk of zoster, improving the uptake of zoster vaccination in this group will also mitigate inequalities in zoster burden.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S10 Table(DOCX)Click here for additional data file.S11 Table(DOCX)Click here for additional data file.S12 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.


We present the first high-quality catalog of early aftershocks of the three mainshocks of the 2016 central Italy Amatrice-Visso-Norcia normal faulting sequence. We located 10,574 manually picked aftershocks with a robust probabilistic, non-linear method achieving a significant improvement in the solution accuracy and magnitude completeness with respect to previous studies. Aftershock distribution and relocated mainshocks give insight into the complex architecture of major causative and subsidiary faults, thus providing crucial constraints on multi-segment rupture models. We document reactivation and kinematic inversion of a WNW-dipping listric structure, referable to the inherited Mts Sibillini Thrust (MST) that controlled segmentation of the causative normal faults. Spatial partitioning of aftershocks evidences that the MST lateral ramp had a dual control on rupture propagation, behaving as a barrier for the Amatrice and Visso mainshocks, and later as an asperity for the Norcia mainshock. We hypothesize that the Visso mainshock re-activated also the deep part of an optimally oriented preexisting thrust. Aftershock patterns reveal that the Amatrice Mw5.4 aftershock and the Norcia mainshock ruptured two distinct antithetic faults 3–4 km apart. Therefore, our results suggest to consider both the MST cross structure and the subsidiary antithetic fault in the finite-fault source modelling of the Norcia earthquake. Among recent earthquakes, the 2010 Mw7.2 El Mayor-Cucapah (Mexico) and the 2016 Mw7.8 Kaikoura  events are illuminating examples of rupture cascades involving distinct fault segments with variable orientations and kinematics4. While there exists a wealth of examples of multi-fault large earthquakes for strike-slip and transpressional settings2, the literature is quite limited for normal-faulting events. The large stress perturbation associated with the 2011 M9.0 Tohoku-Oki mega-thrust earthquake is believed to have induced the sequence of M6-7 normal faulting earthquakes that ruptured complex arrays of fault segments along the Pacific coast of NE Japan5. Extensional provinces affected by multi-fault normal-faulting earthquakes or sequences are: the Basin and Range in western North America6; the Afar in the East Africa Rift zone7; the North Island of New Zealand8; the continental Greece10; the western Cordillera Belt in Spain11; the Apennine seismic belt of Italy12. For the Apennines, multi-fault ruptures during a single seismic event (e.g. 1980 Ms 6.9 Irpinia-Basilicata)13 or sequences spanning a period of days (e.g. 2009 Mw 6.1 L’Aquila)14 to months (e.g. 1997 Mw 6.0 Colfiorito)15, are controlled by the inherent geometrical complexity of the active normal fault systems dissecting a mountain belt mainly made of carbonate and siliciclastic rocks, and characterized by a poly-phased tectonic evolution. Extensional tectonics took place since the Early and Middle Pleistocene in central16 and southern Apennines17, replacing the Neogene compressional tectonics responsible for the thrust-belt accretion and overprinting contractional structures. The average Quaternary extension axis trends NE-SW, mostly parallel to the direction of the orogenic transport, so that several normal faults are nearly coaxial with thrusts. The relative youthfulness of the extension regime and the heterogeneity of the sedimentary multi-layer involved in the orogenic wedge played a key role in the segmentation of the NW-SE trending active normal fault systems16. As a consequence, there is growing evidence that pre-existing compressional structures may control rupture nucleation and propagation during multi-fault normal-faulting earthquakes and seismic sequences. Crustal-scale heterogeneities , arcuate thrust faults and transpressive cross-faults often limit the extent of individual ruptured segments18, while pre-existing thrust faults optimally oriented to slip in the present-day extensional stress field  can be in turn reactivated as normal faults under proper dynamic conditions20. For the Apennines, the extensional reactivation of favorably oriented Pliocene thrust faults is also documented by detailed analysis of fluid-injection induced seismicity21.The key role of geometrical fault complexity in controlling both earthquake nucleation and dynamic rupture propagation is a key issue in the seismic source mechanicsThe Amatrice-Visso-Norcia (AVN) seismic sequence that struck central Apennines with three mainshocks with normal-faulting mechanisms between August and October 2016 poses significant challenges from the perspective of earthquake source characterization and analysis of seismic sequence evolution. In particular, the geometry of the activated normal faults and preexisting compressional structures has emerged as a central element of the scientific discussion about the multi-segment rupture process.23 and was followed on 26 October 2016 by the Mw 5.9 Visso earthquake, ∼25 km to the north  and the Laga Fault system to the south24 Fig. .28. Fault segmentation is ascribed to the inherited Mio-Pliocene Mts Sibillini Thrust . Data were recorded by the INGV National Seismic Network complemented by 12 temporary stations installed soon after the Amatrice earthquake and connected in real-time to improve performance of the monitoring system32. Notwithstanding the good quality of this dataset, the catalog published by22 suffers from implicit drawbacks strictly related to the fact that the analysis procedure at the INGV surveillance center must provide very fast and reliable hypocentral location and local magnitude estimation for civil protection purposes . The location procedure routinely followed during a seismic sequence is based on the revision of P-and S-phase automatic picks provided by the earthquake detection system, complemented by hand-picking of P- and S-arrivals for a limited number of stations surrounding the real-time automatic location. Additionally, in the first days following the AVN mainshocks, seismologists on duty at the surveillance center had to focus just on the strongest aftershocks. Indeed, revision of small early aftershocks detected by the monitoring system posed significant challenges because of the occurrence of very close or even simultaneous events located in different zones of the monitored region.Aftershocks catalog published by22 contains only a subset of the aftershocks detected by the real-time monitoring system, with the number of missed events being larger for early aftershocks, and that events re-location uses only part of the stations and readable arrival times.From the previous considerations, it arises that the catalog published by31, which uses automatic traveltime pickings of ∼44,100 events recorded from 24 August 2016 to June 2017, shows a relatively-low spatial resolution  and variance improvement (32%) in spite of the good station source-receiver geometry, suggesting that uncertainty in the arrival time data degraded the inversion process.The enhanced aftershock production over a wide zone also makes the use of automatic phase picker tools difficult. The local earthquake tomography survey of33. We decided to focus our analysis on early aftershocks for two reasons: first, the number of events detected by the real-time monitoring system but not revised manually was the largest in the first hours after moderate-strong shocks of the sequence; second, accurate locations of early aftershocks can give clues on the initial fault activation and on the geometry and size of mainshock faults providing key information to understand the source rupture processes. The contribution of early aftershocks can be fundamental in case of a multi-fault rupture involving segments with different orientations and kinematics during a single seismic event3. In addition, the anticorrelation between distribution of early aftershocks and main patches of slip can give insight into the rupture process and the pattern of asperities and barriers1.To overcome the previous drawbacks, we performed a careful reprocessing of all the earthquakes detected by the INGV real-time monitoring system in the first days after the Amatrice, Visso and Norcia earthquakes to create the first high-quality and comprehensive catalog of early aftershocks. P- and S-wave first arrivals were hand-picked using all INGV stations installed in the epicentral region and inverted by using a robust, non-linear earthquake location algorithmThe outcome is a high-quality catalog of 10,574 aftershocks characterized by a significant improvement in location accuracy and magnitude completeness with respect to previous studies. The new catalog provides a clearer image of the early aftershock seismicity that improves our understanding of the activated fault systems, thereby highlighting strength and weakness of multi-fault rupture models. In addition, our catalog is valuable for future seismological applications.th–26th (hereinafter early aftershocks of the Amatrice earthquake), October 26th–27th (early aftershocks of the Visso earthquake) and October 30th–November 1st 2016 (early aftershocks of the Norcia earthquake). We handpicked around 291,800 P- and 188,200 S-wave first arrivals of 10,574 aftershocks using 129 permanent and temporary INGV stations installed in the epicentral area  revision of P- and S-phase available data  of aftershocks quickly examined at the INGV surveillance center or simply detected by the real-time monitoring system, (ii) addition of P- and S-phase picks for numerous permanent and temporary stations that were not included in previous automatic or manually revised locations. In addition, our study benefits from data of 9 temporary INGV stations32, which are used for the first time.We analyzed all the aftershocks detected between August 2433.We located events in a 1-D local velocity model by applying the non-linear inversion technique NonLinLocWe computed local magnitude after a careful assessment of the quality of the amplitude measurements, aimed at both correctly managing records affected by the superposition of aftershocks and discarding stations affected by local site effects. Aftershocks have magnitude in the range 0.2–4.7.22, the new catalog shows a significant increase in the number of earthquakes (2.8 to 3.9 times larger depending on the mainshock) and improvement in magnitude completeness (Mc)  ≤0.6 km and ≤2 km, respectively.The distribution of location quality parameters demonstrates the high-quality of the catalog . The strong increase of the phase picks number for most aftershocks causes an important reduction of the azimuthal gap and epicentral distance to the closest station that primarily control horizontal and vertical location uncertainties, respectively  for all events but the Norcia mainshock . The structural architecture of the range is very complex, because it derives from three main extensional and compressive tectonic pulses: (1) pre-orogenic syn-sedimentary extension (Jurassic) controlled the formation of pelagic basin and carbonate platform domains delimited by crustal-scale fault zones; (2) Mio-Pliocene compression affected the previously formed basin and platform domains and was responsible for the accretion of the Umbria-Marche belt structured into NE-verging arcuate thrust faults and associated folds; (3) Quaternary post-orogenic extension accommodated by NNW-SSE trending SW-dipping normal fault systems that dissect the fold-thrust belt and bound large intramontane depressions. The crustal architecture is dominated by the arcuate MST  NNW-trending, SW-dipping event zones related to the causative faults of the three mainshocks, (ii) NNW-trending, NE-dipping event zones referable to antithetic normal faults, (iii) deep seismicity clustering at 8–10 km depth within a band gently dipping eastward.The distribution of early aftershocks and mainshocks, complemented by relocated late aftershocks, yields valuable information on faults activated during the seismic sequence. Coherently withhttp://cnt.rm.ingv.it/tdmt) separately for the three investigated time intervals and compared them to precise surface constraints on Quaternary normal faults41 and Mio-Pliocene thrusts42  inversion of the largest events  asperitiesult Fig. .L < 3 events are located in the top 2 km of the crust in agreement with the lack of evident surface faulting along the Laga Fault system41 and the 2 km thick cover of marly and terrigenous Miocene sediments.Remarkably, the fault plane inferred by TDMT tied to our mainshock hypocenter perfectly fits the eastern splay of the Laga Fault system Fig. . A wide 28 and surface coseismic faulting observed for ∼6 km in strong Mesozoic carbonate rocks39 (maximum surface slip of 0.35 m). The easternmost shallow aftershocks fall in the Vettore Fault footwall delimited by surface ruptures . Their hypocenters projected onto a N110° section  collapse to a WNW-dipping narrow band  and the surface projection of event alignment is compatible with mapped thrust traces  are double earthquakes, both preceded by M39  and finite-fault inversions using InSAR and GPS data . Second, re-activation and inversion of the Pizzo Tre Vescovi thrust are compatible with its geometry optimally oriented in the present stress field that is characterized by NNW-oriented SHmax43.We propose that the Visso earthquake rupture first propagated up-dip onto the deeper portion of the thrust and subsequently onto the shallow and steeper splays of the Cupi fault system, which caused surface faulting  and Fig. Differently from the Amatrice earthquake, early aftershocks of the Visso earthquake did not re-activate the MST. Only a few events fall in the N-trending cloud of off-fault aftershocks of the Amatrice earthquake showed in Fig. 42, we conclude that the thrust illuminated in the foot-wall of the VBFS should have a flat-ramp-flat geometry. The cloud of late aftershocks at the northern edge of the activated zone suggests a structure antithetic to the Cupi fault  and the northern segment of the Laga Fault system, for a total length of ∼35 km Fig. , togetheIn the northernmost sector, the distribution of early aftershocks reproduces that of the Visso earthquake, and suggests re-activation of both Cupi Fault and Pizzo Tre Vescovi thrust Fig. . Convers29, and no clear alignment of early aftershocks appears underneath the Vettore fault. The absence of aftershock alignment might be also an effect of the complexity of the deformation zone that is 2.5 km wide near the Castelluccio basin39.Aftershock production is weak to the east of the mainshock epicenter Figs  and 5e, 29. The strike and length of the antithetic fault inferred from event alignment are ∼N345° and ∼12 km, respectively. Its location is 3–4 km eastward of the antithetic fault activated by the Amatrice Mw5.4 aftershock  of the Umbria-Marche multilayer and the phyllitic upper part of the underlying Permo-Triassic basement. Such deep band of seismicity was ascribed to a regional shear zone and its bottom defines the brittle-ductile transition24.Previous studiesb) is shallower than that of the Amatrice earthquake in the SE sector . A dense cloud of aftershocks of the Amatrice earthquake dips NW delineating the deep part of the lateral ramp of the MST . Remarkably, the bottom of the seismicity band deepens abruptly from 8 km to 11 km depth just at the intersection with this ramp. In addition, the up-thrown sector dips clearly to northwest. All these geometries indicate a crustal-scale contractional nature of the structure illuminated by the NW-dipping cloud of aftershocks  that appears also rooted in the underlying basement, as suggested by aftershocks of the Norcia earthquake  low friction phyllites, (ii) high pore fluid pressure zones just below and/or within the base of the Triassic evaporites, where fluid overpressure can occur within dolostone sealed by anhydrite beds44. Since the deep discontinuity possibly separates normal friction carbonate rocks (μ = 0.6–0.8) and low friction basement phyllites45, it could correspond to a zone of strain rate gradients and stress accumulation where seismicity concentrates.The new accurate hypocentral locations allow investigating the relationship between the deep seismicity and the normal faults ruptured by mainshocks. The activation of the deep band of seismicity follows always the mainshock and its along-strike extent, defined by M ≥ 3 early aftershocks, mimics the total length of the broken fault segments Fig. . In cros24 of a dual role played by the MST lateral ramp in controlling rupture propagation. Early aftershocks of the Amatrice and Visso earthquakes define two distinct contiguous zones of the VBFS separated by the deep part of the MST cross-structure that was illuminated by a wealth of aftershocks of the Amatrice earthquake  dipping MST in the hanging-wall of the Vettore segment, lacks high-angle faults in the footwall of the VBFS and thereby ascribes all eastern aftershock activity to the normal fault system.Our aftershock locations allow placing new geometrical and kinematic constraints on secondary faults. They confirm a SSW orientation for the cross-structure corresponding to the kinematically inverted MST lateral ramp, but also delineate a ramp-flat geometry dipping ∼45° in the upper part. Therefore, our results contrast with the gently-dipping segment of27. Besides, our study unravelling two distinct antithetic structures for the Amatrice and Norcia earthquakes  and shifted (~2 km) to the west. We emphasize that the rupture of the antithetic fault defined in this study during the Norcia mainshock is supported by the good agreement with the location and geometry of the coseismic faulting that affected the San Benedetto freeway tunnel46 22 and Norcia earthquake (9.5 km depth)29, we highlight that our relocated hypocenters better agree with slip patches confined to the top 5–6 km of the crust showed by all rupture models29. In the perspective of a more reliable earthquake source investigation, the improvement in the location of nucleation points of major shocks we have provided in this work should be taken into account in the future finite-fault source modeling attempts.The relatively shallow depth of the three relocated mainshocks  clearly indicates nucleation inside the Umbria-Marche sedimentary multilayer - specifically within the high Vp/Vs zone ascribed to high pore fluid pressure carbonates by47, whereas aftershock activity along the MST lateral ramp supports the rupture model of the Norcia earthquake of29 that requires significant slip on this cross-structure. The spatial partitioning of early aftershocks evidences the dual control played by the MST lateral ramp on the rupture propagation (barrier and asperity) in agreement with interpretation of24. We hypothesize re-activation and kinematic inversion also for the optimally oriented Pizzo Tre Vescovi thrust whose deep part was likely ruptured by the Visso earthquake. Two distinct main antithetic faults ruptured during the August 24 Mw5.4 aftershock and the Norcia mainshock, respectively. Therefore, our study suggests to consider both the MST cross-structure and the eastern antithetic fault in future finite-fault source modeling of the Norcia earthquake that hopefully should combine strong motion, GPS and InSAR data.Aftershock patterns illustrated in this study highlight the complex interaction between normal-fault systems and inherited thrusts activated during the AVN sequence and provide new insight into the multi-segment rupture mechanisms. We document reactivation of a flat-ramp structure referable to the MST. Both its N-trending frontal ramp in the VBFS footwall and its SSW-trending lateral ramp in the Vettore fault hangingwall are prolific in aftershock production. The off-fault aftershock cloud that elongates N-S following the MST frontal ramp can be explained by the Coulomb static stress changes induced by the Amatrice earthquake48 and for optimizing automated picking algorithms49. Then, application of high-precision double-difference relative location techniques strictly depends on the quality of the earthquake catalog and on the accuracy of initial absolute locations50. Future finite-fault inversions will benefit from geometric constraints on the activated fault systems and mainshock locations provided by our study. The new high-quality hypocentral locations will allow improving comparison between structures imaged by seismic reflection data and aftershock distribution.Our high-quality manually reviewed catalog is of key importance for future seismological applications. For a prolific aftershock sequence like AVN, a high-quality initial catalog with a wide range of magnitude is a key ingredient for the successful application of detection techniques based on template waveforms cross-correlation33, which is based on the probabilistic approach proposed by51 and on a robust non-linear inversion scheme. NLL offers the advantage to provide a comprehensive estimate of the location uncertainties through the construction of the a posteriori probability density function (PDF). Following the probabilistic approach, the optimal earthquake hypocenter location is represented by the maximum likelihood point of the computed PDF, while the shape and the size of the PDF are representative of the location uncertainty .We located both aftershocks and mainshocks using the inversion program NonLinLoc 2, centered on the epicenter of the Norcia mainshock (Fig. 52 (Fig. The NLL inversion grid extends over an area of 120 × 120 km 52 Fig. . StationSupplementary MaterialsSupplementary Video S1Supplementary Video S2Supplementary Video S3Data S1


Tele-Savvy is a 3-arm randomized controlled trial (RCT) of a psychoeducation intervention that equips dementia family caregivers with the knowledge and skills they need to provide care to their person, while also caring for themselves. This RCT is currently underway, with cohorts rotating through over a period of 12 months. The purpose of this presentation is to explore the effectiveness of Tele-Savvy (active) versus Healthy Living Intervention (attention control) or usual care (waitlist) on self-care behaviors among dementia family caregivers. We conducted semi-structured interviews with 16 caregivers after their initial participation in either the active, attention control, or usual care groups. Interviews elicited caregivers’ perceptions regarding the program’s influence on their self-care behaviors and engagement in self-care activities. The overall emerging theme for the family caregivers who participated in the Tele-Savvy and Healthy Living programs was “increased awareness of self-care activities”. Family caregivers in the Healthy Living program spoke mainly about engaging more in physical activities and improving their nutrition by eating healthier and keeping track of foods. Across all study groups, social engagement and having a support system were common self-care activities. Various limitations to engaging in self-care activities while in enrolled in the study included time and caregiving responsibilities. Suggestions were made for more respite services to become available to allow for family caregivers to engage in self-care activities. These findings provide researchers and practitioners with pertinent information to develop and refine programs for family caregivers to improve their self-care practices.


This study aimed to analyse the patient predictors of health-seeking behaviour for persons coughing for more than 2 weeks to better understand this vulnerable and important population.The study analysed data from a cohort study (SOCS - Secondary Outcome Cohort Study) embedded in a community randomised trial ZAMSTAR (Zambia and South Africa TB and AIDS Reduction Study) in eight high-burden TB communities in the Western Cape, South Africa. These datasets are unique as they contain TB-related data as well as data on health, health-seeking behaviour, lifestyle choices, employment, socio-economic status, education and stigma. We use uni- and multivariate logistic regressions to estimate the odds ratios of consulting for a cough (of more than 2 weeks duration) for a range of relevant patient predictors.. In the multivariate analysis, respondents of black ethnic origin , those with higher levels of education , and older respondents  had a higher likelihood of consulting for their chronic cough. Individuals who smoked  and those with higher levels of socio-economic status  were less likely to consult. We find no evidence of stigma playing a role in health-seeking decisions, but caution that this may be due to the difficulty of accurately and reliably capturing stigma due to, amongst other factors, social desirability bias.Three hundred and forty persons consulted someone about their cough and this represents 37% of the 922 participants who reported coughing for more than 2 weeksThe low levels of consultation for a cough of more than 2 weeks suggest that there are opportunities to improve case-finding. These findings on health-seeking behaviour can assist policymakers in designing TB screening and active case-finding interventions that are targeted to the characteristics of those with a chronic cough who do not seek care.The online version of this article (10.1186/s12913-019-3992-6) contains supplementary material, which is available to authorized users. TB transmission is amplified in the context of the prevailing HIV pandemic in the WHO  African region, where 31% of all new TB cases in adults are attributable to HIV infection , 3. ThisDelays in seeking healthcare for a cough may arise from the patient or the healthcare system, or, most likely, both. The focus of this study is on understanding how individual characteristics are correlated with the health-seeking decisions of symptomatic cases.Very few studies comprehensively investigate health-seeking behaviours from the patient perspective exclusively , 7. MoreThe most recent systematic review of delays in TB diagnosis found that evidence describing the potential risk factors for delay are heterogeneous . A risk Responding to the global call to address stigma in the fight against TB , 14, thiThis cross-sectional study uses SOCS and other ZAMSTAR data to estimate the odds ratios of consulting for a cough (of more than 2 weeks) for relevant patient predictors.ZAMSTAR was a community-randomised trial that aimed to reduce TB and AIDS in South Africa and Zambia, two countries with amongst the highest TB incidence rates globally (in 2010 TB incidence in Zambia was 495/100000 people and 948/100000 for South Africa ), using The aim of the SOCS was to recruit TB-affected households in each of the ZAMSTAR communities in order to collect stigma-related data from confirmed TB patients and their family members . This stn = 30,017) in South Africa that provides such a comprehensive range of variables at an individual, household and community level.These datasets – ZAMSTAR and SOCS - are unique given that they contain TB-related data as well as data on health, health-seeking behaviour, lifestyle choices, employment, socio-economic status, education and stigma. There is currently no other TB-related dataset of this size  were included as continuous covariates.Multiple Correspondence Analysis and Related Methods [The asset index was derived from household asset ownership indicators using multiple correspondence analysis (MCA) , access to electricity, dwelling type , domestic assistance, main type of toilet (including flush toilet and bucket system), main source of drinking water (including piped sources and wells), fuel access for keeping warm (including electricity and wood) and reliance on food relief  assets indicate a higher SES . A proxy for HIV was included as a dichotomous health-status covariate.Lifestyle-choice binary variables related to smoking and drinking  were included. These covariates were the only lifestyle-choice variables available in the ZAMSTAR dataset.Respondents of the SOCS survey were presented with questions or statements linked to seven TB-stigma domains: unnecessary fears of transmission, blame, experience of social exclusion, experience of being made fun of, experience of health-setting stigma, internal stigma and disclosure  and a chi-square test (for dichotomous covariates) were used to test for significant differences between the two sub-groups.A logistic regression was used for uni- and multivariate analyses. In the model, the dependent variable captures health-seeking behaviour and relevant independent variables include socio-economic status, demographic, health status, lifestyle choice and stigma.P = 0.18).In line with best practice , a regreEmpirical analyses were performed using STATA .Three per cent of the full ZAMSTAR sample  reported coughing for more than 2 weeks. These 922 observations constitute the sample of interest. Thirty-seven per cent (340 persons) of this sample consulted someone about their cough, while the majority - 63% (582 persons) - did not.The summary statistics of the sample characteristics are described below Table .Table 1SThe mean age of those who consulted for their 2-week cough was slightly higher; 42 years compared to those who did not consult (39 years). The mean highest level of education for those who consulted and those who did not was the same – namely Grade 8 (8 years of education).The asset index ranged from − 3.08 to 1.20 with a larger index representing a higher level of SES. The negative relationship between the probability of consulting and the asset index .Twenty-two per cent of those who consulted were HIV positive, while 17% of those who did not consult were HIV positive  than amongst those who did consult (36%).The stigma index ranged from − 1.73 (minimum) to 1.78 (maximum) with a larger index representing a higher level of stigma. For those who consulted, the mean stigma level was significantly lower (− 0.12) compared to those who did not (− 0.03).P-values for the univariate regression are described first, followed by the results for the multivariate regression.Results for the univariate and multivariate logistic regressions Table  are repoStigma has a statistically insignificant relationship with the probability of seeking healthcare for a cough of more than 2 weeks . The stigma result remained insignificant  even when the multivariate regression was rerun without the variables for SES and ethnicity.men consulting is less than one in the univariate regression  but this becomes statistically insignificant in the multivariate regression . Employment does not show any significant relationship with the likelihood of consulting though the likelihood of the employed consulting is consistently less than the unemployed . Being of black ethnic origin and a year older is associated with a higher likelihood of consulting . In the case of respondents of black ethnic origin, the reference case is respondents of Cape coloured ethnic origin.The odds ratio of asset index was less than one . For each additional year of education, the odds of consulting was 1.05 times more likely in the multivariate results .The odds ratio of consulting for each standard deviation increase in the HIV-positive individuals were more likely to consult than their HIV-negative counterparts or those who did not disclose their HIV status  but these findings are not statistically significant in the multivariate regression. There was no significant increased likelihood of consulting for drinking daily . Persons having smoked were significantly less likely to consult .P < 0.01), those with higher levels of education , and older respondents  had a higher likelihood of consulting for a chronic cough. The opposite held for those who smoked  and those with higher levels of socio-economic status .Only 37 % of respondents had consulted someone when coughing for more than 2 weeks. Respondents of black ethnic origin  and found that at older ages this relationship persists, which is in contrast with the health-seeking literature [Cultural dynamics not fully captured in the stigma variable may explain the relatively large, statistically significant differential in the relationship between ethnicity and health-seeking behaviour. Confirming previous findings in the health-seeking literature , we findterature .The positive association between HIV and consulting is not significant, but this may be due to an under-reporting of HIV positive cases.Having smoked is the only significant lifestyle choice variable, with those having smoked being less likely to consult. It is possible that those who have smoked attribute their symptoms to smoking rather than TB . This reExternal validity is limited to high TB-prevalence communities in South Africa but given South Africa’s high TB prevalence and incidence, these findings are still of international interest. Finding no role for stigma in health-seeking behaviour is important because it highlights the need to triangulate methods and debate how stigma measures interact with other factors in multivariate analyses.Most persons coughing for more than 2 weeks did not seek consultation about their chronic cough. Respondents of black ethnic origin, those with higher levels of education and older respondents had a greater likelihood of consulting. Smokers and those with higher SES were less likely to consult.TB-control programmes should be more cognisant of the patient characteristics of presumptive TB patients who do not seek care. This information can assist policymakers in designing more targeted TB screening and active case-finding interventions. In the high TB burden communities of South Africa, such interventions may benefit from focusing on youth, those with lower levels of education, smokers, higher SES subgroups and also the population of Cape coloured ethnic origin. These findings add to the meagre body of literature analysing the health-seeking behaviour of this vulnerable and important population.Our study responds to the global call to address stigma in the fight against TB by being the first (as far as we know) to include a quantitative measure of TB stigma in modelling health-seeking behaviour. Our uniquely comprehensive data set allows us to address this gap in patient-focused analysis of health-seeking decisions. Although we find no role for stigma in health-seeking behaviour, it is important because it highlights the need to triangulate methods and debate how stigma measures interact with other factors.Additional file 1:SES indicator set. Table showing SES components collected in the ZAMSTAR study that was used to create an asset index for this study. (DOCX 13 kb)Additional file 2:Correlation between components of asset index and asset index. Table showing correlation between components of asset index and asset index. (DOCX 14 kb)Additional file 3:TB-stigma indicator set. Table showing TB-stigma indicator set used in the SOCS study. (DOCX 13 kb)Additional file 4:Stigma level by community (a), age (b) and gender (c). Figures (a-c) showing mean stigma index levels (standardised using z-score) for each community (1–8), for each age category  and for each gender (women and men). Source: own calculations, SOCS of ZAMSTAR (2008/2009). (DOCX 26 kb)


Chronic pain is among the most burdensome conditions. Its prevalence ranges between 12% and 30% in Europe, with an estimated 21% among Austrian adults. The economic impact of chronic pain from a societal perspective, however, has not been sufficiently researched. This study aims to provide an estimate of the societal costs for working-age adults with chronic pain in Austria. It explores the impact of sex, number of pain sites, self-reported pain severity, health literacy and private health insurance on costs associated with chronic pain.A bottom-up cost-of-illness study was conducted based on data collected from 54 adult patients with chronic pain at three Viennese hospital outpatient departments. Information on healthcare costs including out-of-pocket expenses and productivity losses due to absenteeism and informal care were collected over 12 months. Resource use estimates were combined with unit costs and mean costs per patient were calculated in € for year 2016.Mean annual societal costs were estimated at EUR 10191. Direct medical costs were EUR 5725 including EUR 1799 out-of-pocket expenses (mainly pain relieving activities and private therapy). Productivity losses including informal care amounted to EUR 4466. Total costs for women and patients with three or more pain sites were significantly higher. No association with health literacy was found but there was a tendency towards higher out-of-pocket expenses for patients with complementary private health insurance.This study is the first to provide a comprehensive assessment of the individual and societal burden of chronic pain in Austria. It highlights that chronic pain is associated with substantial direct medical costs and productivity losses. Patient costs may show systematic differences by health insurance status, implying a need for future research in this area. Chronic pain is considered as one of the most burdensome diseases in industrialized countries . AccordiIn the same study, prevalence of chronic pain was estimated at 21% for Austria, amounting to 1.83 million people in 2003 . AccordiTo assess the societal and economic impact of chronic pain, several cost-of-illness studies have been conducted across Europe e.g. –17). Dif. Dif17])Regarding drivers for differences in the costs of chronic pain, several studies have investigated associations between demographic factors, socioeconomic characteristics and disease severity with cost levels e.g. , 23–25) –25 23–25The aim of this cost-of-illness study was to estimate the economic burden of chronic pain in working-age individuals from the health care, patient and societal perspectives. The impact of sex, number of pain sites, self-reported pain severity, health literacy and complementary private health insurance status on costs associated with chronic pain were also explored. Such a cost-of-illness analysis allows assessing the overall economic impact of chronic pain and its main cost components, aids the priority setting for health care policies and may inform future resource allocation , 28.Patient recruitment took place between December 2012 and February 2014 as part of the study ‘Chronic pain and its development depending on social environment, health literacy, and previous treatment and associated costs’. Participants were recruited from three hospital outpatient departments  located at two  ViennesFollowing a medical check, participants were informed about the study. After signing the informed consent form, participants completed the baseline questionnaire with the help of a research assistant. The baseline questionnaire captured socio-demographic details including sex, age, relationship status, employment status, highest educational attainment, monthly household income (after taxes), complementary private health insurance and exemption from prescription fees. The location and derived number of pain sites was assessed based on a pictorial representation. The number of pain sites was categorized into ‘1 or 2 pain sites’ and ‘3 or more pain sites’ and served as a proxy for the widespread nature of the pain. A visual analogue scale (VAS) ranging from 0  to 100 (‘the worst possible pain’) captured the subjective pain intensity and was categorized into high self-reported pain severity (VAS>36) and low pain severity (VAS≤36) based on the observed mean . PolyphaA cost diary was used to capture disease-attributable resource use within the past three months. It was informed by previous research  and piloTotal costs were calculated from a societal perspective following a bottom-up approach . Direct Resource use data were multiplied with unit cost estimates to calculate costs. Unit costs were retrieved from a number of sources  based onBased on the information provided in the four cost diaries, costs were calculated for 12 months. In the case of missing cost diaries, we assumed that costs described in 3, 6 or 9 months were representative for the missing months and reported them for a 12-month period. All costs were calculated in Euro for year 2016 to reflect the latest available unit cost information at the time of manuscript submission. Cost data from previous years were inflated based on the national medical component of the consumer price index .The database was initially set-up in Microsoft Excel (2013) and double-checked by two authors . Most cost diaries were fully completed. Missing items only accounted for 0.5% of all data entries and were replaced with mean values from all patients with relevant resource use. As some patients dropped out during the study, the cost data of these participants were extrapolated proportionately based on their available cost diaries. A sensitivity analysis restricted to patients with full cost information over the 12 months follow-up period was also conducted.All statistical analyses were performed using Stata 14 . DescripThe study was approved by the Ethics Committee of the Medical University of Vienna  for the General Hospital of Vienna and the municipality of Vienna  for the Orthopedic Hospital Speising. While advance information of potential study participants was started in December 2012, no primary data were collected prior to receiving formal ethical approval.Out of a total of 121 participants initially enrolled in the chronic pain study, 54 participants had at least 3 months of cost data  and were included in the cost-of-illness analysis . Thirty-three patients had full 12 months cost data and were included in the sensitivity analysis. With one exception, no differences in demographic, socio-economic and clinical characteristics  were found between all initially enrolled patients (n = 121), the responders (n = 54) and the patients with 12 months cost data (n = 33). A higher percentage of the initially enrolled patients  reported living in a partnership compared to the 54 patients  included in the main analysis (p = 0.03). In total, 78% of the study sample were female (n = 42) . Men andOn average, patients were aged 50 (SD: 8.0) years, 69% (n = 37) worked and 63% (n = 34) lived in a partnership . Fifty-sDetails of the mean annual resource use are provided in Out of the 54 patients, 33 (61%) reported having taken at least one prescription medication over the last 12 months  and 15% From the viewpoint of the patient, pain reducing lifestyle activities that prevent or alleviate chronic pain (e.g. sports like swimming and yoga) , private therapist costs  and household help  were the dominant contributors to out-of-pocket expenses . A totalOverall, productivity losses  contributed to almost half of the total costs , Fig 1. Mean total annual cost per chronic pain patient were estimated at EUR 10191  . In the Total costs for men and women differed significantly  and were estimated at EUR 3095 (SD: 2709) and EUR 12219 (SD: 13422), respectively . Mean toThis cost-of-illness study aimed to comprehensively assess the economic burden of chronic pain in the working-age population in Austria. Based on a sample of patients recruited from three hospital outpatient departments, annual societal costs were estimated at EUR 10191 per patient for the year 2016. The findings emphasize the high economic impact of chronic pain on both the individuals and the society. The highest direct medical costs were associated with out-of-pocket expenses (18%), inpatient costs (14%) and outpatient costs (8%). Highest productivity losses were associated with early retirement/work reductions (16%) and sick leave (14%). In terms of potential drivers for differences in chronic pain costs, women and patients with three or more pain sites had significantly higher total costs than men and patients with one or two pain sites. No association between costs and health literacy or self-reported pain severity was found. There was a tendency towards higher out-of-pocket expenses for chronic pain patients with a complementary private health insurance.In comparison with another Austrian study measuring the direct costs of chronic low back pain in 2008 (EUR 1837 in 2008) , total dOn the other hand, the patterns in costs components found in our cost-of-illness analysis are consistent with other national and international studies. Out-of-pocket expenses were similar to the out-of-pocket expenses of Dutch chronic pain patients USD 1350 in 1999) . The ten0 in 1999In terms of differences in chronic pain costs by subgroups, female sex was found to be a major determinant of cost differences. While this result is in line with some previous work , 23, it The identified tendency for higher out-of-pocket expenses for people with complementary private health insurance  needs to be interpreted in the light of the Austrian two-tier health system. While private health insurance status is likely to be also related to higher socioeconomic status , it is aTo our knowledge, this is the first study to estimate the societal burden of chronic pain in Austria. In addition, this is one of the few studies internationally that measures related societal costs comprehensively. Besides investigating commonly researched associations between costs and demographic and clinical characteristics, this study also adds to the literature through the analyses of the impact of health literacy and complementary health insurance on the costs of chronic pain.The findings of this cost-of-illness analysis need to be interpreted in the health care system context and considering potential limitations. The small study sample and recruitment from three outpatient clinics in the capital might impair the generalizability of the results to the national level. Firstly, with an average of 17 years, the duration of chronic pain was longer than the (more conservative) general Austrian estimate of 6 years from 2003 . AnotherThe costs of chronic pain including patients’ out-of-pocket costs were found to be substantial in Austria and the trend towards systematic differences by health insurance status calls for future research in this area. The comparatively low proportion of patients contributing to the high productivity losses illustrates the need to emphasize and strengthen early diagnosis, prevention and better management of chronic pain as a priority from a public health policy perspective. More effective and efficient treatment programs–even if these treatment costs are high–could potentially reduce early retirement and sick days and lead to a good value policy option with overall lower cost-of-illness in Austria.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S3 Text(DOCX)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


Hematopoietic stem cells (HSCs) play an important physiological role as regulators of all blood and immune cell populations, and are of clinical importance for bone marrow transplants. Regulating HSC biology in vitro for clinical applications requires improved understanding of biological inducers of HSC lineage specification. A significant challenge for controlled HSC expansion and differentiation is the complex network of molecular crosstalk between multiple bone marrow niche components influencing HSC biology. We describe a biology-driven computational approach to model cell kinetics in vitro to gain new insight regarding culture conditions and intercellular signaling networks. We further investigate the balance between self-renewal and differentiation that drives early and late hematopoietic progenitor populations. We demonstrate that changing the feedback driven by cell-secreted biomolecules alters lineage specification in early progenitor populations. Using a first order deterministic model, we are able to predict the impact of media change frequency on cell kinetics, as well as distinctions between primitive long-term HSCs and differentiated myeloid progenitors. Integrating the computational model and sensitivity analyses we identify critical culture parameters for regulating HSC proliferation and myeloid lineage specification. Our analysis suggests that accurately modeling the kinetics of hematopoietic sub-populations in vitro requires direct contributions from early progenitor differentiation along with the more traditionally considered intermediary oligopotent progenitors. While consistent with recent in vivo results, this work suggests the need to revise our perspective on HSC lineage engineering in vitro for expansion of discrete hematopoietic populations. Hematopoietic stem cells (HSC) mediate production of the body’s full complement of blood and immune cells through a series of linked differentiation steps. HSCs are primarily located in distinct niche microenvironments in the bone marrow where their activity is strongly influenced by a constellation of molecular signals from local niche components including other niche cells, extracellular matrix (ECM), and soluble or ECM-immobilized biomolecular signals, 2. Prim+ HSCs from a donor ) are positive, the self-renewing fraction of any cell type fi is always ≤ fi max.Progenitor populations have the ability to self-renew and the fraction of self-renewing progenitors at any given time .A quintessential property of stem cells is their ability to be in a quiescent state, where they are not involved in any biological activity such as proliferation or differentiation. The LSK state was assigned a quiescent fraction i to cell state j is given by DRitoj (#cells/cell/day) and is a constant.The differentiation rate for cell state j. This dynamic is dictated by feedback provided by the Terminal cells via concentration of the secreted differentiation stimulators: c3*#Terminal cells. This active concentration is denoted Td, such that:jmax (≤ 1) and is a constant. Increasing numbers of Terminal cell population trigger a negative feedback response within the LSK differentiation dynamic that drops the differentiating fraction (j). Therefore, as [Td] increases, Finally, we assign a fraction to LSK cells that transition directly into the Terminal state, while skipping CMPs as The computational model for the 5-state system was similar to the 3-state model with the following exceptions:Proliferation rates for each cell type was a constantCell secretion rates were constantjST-HSCtoTerminal and jMPPtoTerminalJump fractions were assigned to 2 different cell states: qLT-HSC, qST-HSC, qMPPQuiescent fractions were assigned to 3 different cell states: The full system of differential equations developed based on these criteria for both 3-state and 5-state models, along with parameter values are provided in the S1 file.−cKit+ Sca1+) cells, late progenitors (common myeloid progenitors (CMP: Lin–c-kit+Sca1–CD34+CD16/32–), and terminally differentiated  [Fig 1C–1E). While the Terminals cells reached a peak population of 6 x 105, LSK cells oscillated in number between 102 and 2 x 103, and CMPs maintained a variable but overall low numbers (<300) throughout. The relative numbers and time-scales of these responses highlight that hematopoietic sub-populations may display vastly different kinetics despite being in the same culture.The classical model of HSC hierarchy involves stepwise differentiation of early long-term repopulating progenitors into late oligopotent progenitors and eventually terminally differentiated mature myeloid or lymphoid cells . Primary and anal. Cell-secreted biomolecules that directly or indirectly influence cell kinetics such as proliferation, apoptosis, and differentiation were categorized into 4 types: differentiation stimulators (DiffS) and inhibitors (DiffI), and proliferation stimulators (ProS) and inhibitors (ProI). Cell secretion rates are designed to dynamically respond to active biomolecule concentrations through a feedback mechanism such that the culture system, and the model, naturally adjusts to temporal changes expected due to cell secretions and cell-cell interactions. We expressed the temporal changes in kinetic parameters such as the self-renewal fractions , quiescent fraction of the early progenitors (qLSK), and cell proliferation rates  as functions of biomolecule concentrations. Given recent evidence [jLSK) circumvent the intermediate CMP state and differentiate directly into Terminal cells, making the CMP state more transient in the developmental process of mature myeloid phenotypes.We developed a first order differential equation model describing the kinetics of growth and early myeloid specification for a 3-cell state model of early hematopoietic differentiation evidence  for a twFig 2B–2D). Interestingly, the model predicted the existence of an early spike in the CMP population prior to day 1 of culture . Given the high correlation between model fit and experimental data, we considered the possibility that the peak was not a model anomaly but existed at a hitherto untested data point. We verified the existence of this peak by repeating experiments to gather data points both before (12h) and after (2 days) the original day 1 data point . Excitingly, the model successfully predicted the peak increase for the CMP sub-population prior to the day 1 experimental data point, while accurately representing the kinetics of LSK and Terminal cells throughout the 12h to 2-day culture period. Notably, the presence of a jump fraction (jLSK) was crucial in obtaining a high degree of correlation between model predictions and experimental data. Eliminating this fraction and its corresponding kinetics led to a poor experimental-model fit . This suggests a more involved differentiation process than the traditionally accepted stepwise differentiation pathway for mature cell development. While even a relatively simple HSC liquid culture displays complex cell growth profiles, the mathematical model was able to robustly capture key features of its dynamics and nuances.Model simulations were able to successfully capture changes in cell numbers for all 3-cell sub-populations, providing a strong validation for the underlying kinetics described , presumably with decreased differentiation and increased proliferative rates. Not surprisingly, the number of quiescent LSKs remain relatively low while a steady fraction of LSKs continue transitioning into Terminal cells over time. The importance of cell-secreted biomolecules in regulating feedback-mediated cell kinetics is observed through their respective profiles . Higher concentrations of predicted differentiation stimulators during early culture periods promote Terminal cell expansion, while the increase in differentiation inhibitors secreted by the mature progeny contribute to the observed increase in progenitor numbers. Interestingly, the inflection point for this change is around day 4 and corresponds to the changes in cell profiles. The proliferation rates for each cell type remain relatively constant through most of the 9-day culture period , while the increase in apoptosis rates at longer time points correlate with a decrease in SCF and nutrient availability owing to higher cell numbers .We established temporal profiles of kinetic parameters involved in cell proliferation and differentiation. Here, the model suggests the self-renewing fractions of LSK and CMP increase with longer culture periods  and MPP (jMPPtoTerm) cells transitioning directly into Terminal cells . Based on kinetic profiles of the 3-cell model, proliferation, apoptosis, and cell secretion rates were set constant for all cell types. We observe that the LT-HSC population reduces significantly over the culture period while the MPP and CMP fractions have similar growth kinetics. The bulk of the early progenitor activity was compartmentalized in the ST-HSC sub-populations, where these cells showed biphasic behavior similar to that observed for the larger LSK fraction in the 3-state model . The 5-state model was able to successfully fit the growth curves for all the five cell types simultaneously . The model predicts that similar to the 3-state model the jump fractions remain relatively constant over the 9-day culture period and with roughly equal contributions from ST-HSCs and MPPs . The model also predicts a high fraction of LT-HSCs remain quiescent while ST-HSCs show elevated self-renewing fraction in culture . Similar to the 3-state model, the presence of jump fractions was critical for a high degree of model-data fit. Model simulations failed to capture the growth kinetics of all cell populations, over-estimating the number of early progenitors due to the absente jump fractions, while underestimating the Terminal cell populations possibly owing to delayed differentiation .Building upon the 3-state system, we increased the model complexity by separating the heterogeneous LSK population into 3 discrete stages: the most primitive long-term repopulating HSC (LT-HSC), the short-term repopulating HSC (ST-HSC), and the multipotent progenitors (MPP)  , 29. Ouri (cell states) x j (parameters) matrix for S values, which were then projected onto a heat map, developed for both the 3-state  and 5-state model . Overall, the Terminal cell populations were the least sensitive to parameter perturbation for both model structures, possibly on account of their large numbers and cellular heterogeneity that was not accounted for in the model. The most number of high-impact parameters were observed for ST-HSCs, MPPs, and CMPs, that possessed both a dynamic growth profile and were the early drivers of cell growth in culture. Kinetic parameters such as maximum proliferation rates , differentiation rates, self-renewing fractions (fST-HSC-max), and select secretion constants had a large impact on system response. Interestingly, the impact of these parameters was consistently observed across all cell populations. The 3-cell model elicited a similar parameter sensitivity response with consistencies observed for both LSK and CMP populations.System stability in response to perturbations in individual parameter values is an essential part of the model and presents an opportunity to identify critical parameters to exploit in future experimental cultures. From an engineering perspective focused on developing a more guided HSC culture platform, we endeavored to identify biological or kinetic parameters that have the greatest impact on each cell state. Local parameter sensitivity analysis was performed by defining the sensitivity S where :S=〈ΔO/OS8 Fig) highlight the highly complex system response underway, as can be seen from the non-linear profiles across all parameters and cell populations. Interestingly, inflection points for all cell types are consistently observed around days 3–5, where the corresponding cell growth kinetics are the most volatile. These data suggest that accurate experimental determination of high-impact parameters is vital to developing an accurate and stable model. Sensitivity information of this nature can be potentially utilized to dynamically alter targeted parameters (based on high sensitivity) at specific time points  to modulate cell fate.To address our long-term goal of developing engineering system to selectively expand more primitive HSC populations in vitro, we closely examined cell-specific sensitivities in order to identify the temporal trends in parameter sensitivities. The sensitivity profile varied widely across all conditions, with sensitivities either increasing, decreasing, or changing dynamically over time. Increasing parameter sensitivities are an indication that model stability is weakened over longer culture periods. Representative profiles of parameter sensitivities  Lin−cells progenitor cell fate is directly integrated with cell-cell signaling mechanisms and cell-secreted biomolecules [Csaszar et al. employed an integrated computational and experimental approach to increase the yield of HSCs within a closed culture system by regulating the negative paracrine signaling feedback via a controlled media dilution method [in vitro. The role of inhibitory factors secreted by differentiating hematopoietic cell populations on progenitor cell differentiation and proliferation arrest has been previously observed [Marciniak-Czochra et al. proposed parallel multi-compartment models for HSC differentiation to examine the relationship between stem cell self-renewal and proliferation rate and the expansion of progenitor populations. Their results elegantly identify the role of early progenitor population self-renewal in steady state hematopoiesis [The ability to identify key parameters that dominate culture kinetics and utilizing them to engineer optimal conditions for stem cell expansion is important for future advances in hematopoietic stem cell biomanufacturing. Bone marrow transplants that rely on high numbers of repopulating hematopoietic stem and progenitor cells for marrow reconstitution could benefit from such strategic cell expansion. Our efforts build on key findings in the literature. Using a mathematical model, olecules , 21. Csan method . Buildinobserved . Marciniopoiesis . NotablyNotta et al. therefore proposed a two-tier hierarchy of hematopoietic development in adult hematopoiesis[S10 Fig). It is important to note that all cell populations are detected via surface antigen expression profiles at discrete time points. Consequently, nuances of single cell differentiation process may diverge due to transience of gene or surface antigen expression patterns. It is likely that understanding more deeply population dynamics of cohorts of hematopoietic cells, classified via a series of constants in this model, may involve more complex culture kinetics. Therefore, a more deterministic partial differentiation equation model (PDE) may be required for future efforts to gain more comprehensive understanding of the entire HSC differentiation hierarchy. Such models have been employed to investigate the role of cytokine signaling on cancer cell activity[The concept of a non-hierarchical, discrete development of lymphoid and myeloid progenitors through HSC differentiation has been gaining traction. The existence of the proposed adult lymphoid-primed multipotent progenitors (LMPP) that lack erythroid and megakaryocyte lineage specification question the hierarchical relevance of common myeloid (CMP) and lymphoid (CLP) progenitors in the classical HSC differentiation model, 32. Nottopoiesis where th activity, cell cy activity, and the activity. Interes activity–38. So w activity, suggestS7 Fig). We assessed parameter impact roughly split into an early and late-stage response in culture. A rapid initial response implies that a higher degree of control during the early culture phase is important for regulating the final outcome. Experimental determination of some of the unknown parameters could potentially improve model stability and provide a more reliable model construct. While the differentiation rates in the model are assumed constant over the 9-day culture period, the dynamic cellular feedback observed hints to a more non-linear and interdependent rate kinetics. A more stable model allows us to better establish such kinetics and enable a model-parameter system capable of accurately predicting cell behavior for a wider range of culture conditions. Although the mathematical expressions and the number of parameters used in our model can be subject to modification, the approach described here highlights the possibility of describing complicated cell culture dynamics with a high degree of accuracy. A reduction in the number of variables would reduce model complexity, but at the cost of kinetic information such as temporal shifts in proliferation or apoptosis rates, dynamically changing self-renewal vs. quiescent fractions, and signaling cascades between cell populations. The dependence of cell response on biomolecular feedback identified by this model presents an opportunity to tailor culture systems response in a manner similar to that exploited to create bioreactor paradigms for HSC culture [Sensitivity analysis provides key insight into model stability and identification of parameters that have the biggest influence on culture kinetics. They represent master descriptors of optimal culture conditions with stem cell biomanufacturing potential. The parameter sensitivities observed are an indication of model stability; while low sensitivity values indicate minimal response to system perturbations the retained parameters remain crucial to model output. Our model is sensitive to many parameters at varying degrees, the most prominent being the proliferation and differentiation rates of individual cell populations . Such tools will likely require the addition of supporting niche cells, exogenous biomolecules, and the presence of a biomaterial matrix , 41 within vitro culture kinetics of murine hematopoietic stem cells. Used as a prognostic tool, the model was able to capture the multi-scale population shifts in multiple hematopoietic subpopulations and predict cell response to different culture conditions in a means that could be used to tailor experimental conditions for selective expansion of discrete hematopoietic sub-populations. We demonstrate that the CMP population represents a transient state within the HSC hierarchy and much of the downstream differentiation is driven by rapid differentiation of primarily short term repopulating HSC progenitor populations. These observations are consistent with the recently proposed tiered rather than step-wise HSC differentiation model. Finally, via sensitivity analyses we identify culture parameters, such as proliferation and biomolecule secretion rates, most strongly associated with the observed experimental responses as a means to generate more complex HSC culture models optimized for targeted cell expansion.We employ an integrated computational and experimental approach to successfully describe S1 File(DOCX)Click here for additional data file.S1 Fig–) cells, followed by gating into Lin−(FITC) cells. The LSK population is gated across c-kit+ (PE) and Sca1– (APC-Cy7) and a Pre-CMP selection is made for the c-kit+ and Sca1–, followed by the CMP gate Lin–ckit+Sca1–. The LSK population is further gated into LT-HSC (Flk2– CD34–), ST-HSC (Flk2– CD34+) and MPP (Flk2+ CD34+). (B) Representative FACS plots of the early progenitor populations across 9 days in culture, with the fractions denoting their relative populations from the LSK sub fraction. Overall, the ST-HSC population increases most substantially whereas the size of LT-HSC and MPP populations remain small throughout.(A) Singlets selected from the population are first gated into live (DAPI(TIF)Click here for additional data file.S2 FigjLSK value is set to 0, the model is unable to capture the experimental data for (A) LSK, (B) CMP, and (C) Terminal cells, particularly at long time periods. The lack of early jump fraction leads to a predicted over-accumulation of the CMP cells and reduced size of the Terminal cell population. Error bars represent standard error of mean.When the (TIFF)Click here for additional data file.S3 FigfLSK) and CMP (fCMP) cells, the fraction of LSKs that ‘jump’ to Terminal cells while bypassing the CMP stage (j) and the relatively low quiescent numbers of the early progenitors (q) in culture. A relatively high fraction of LSK cells transition into Terminal cells even during day 9 of culture.  The concentrations of proliferation and differentiation stimulators and inhibitors in the culture media dictate the shift of cell fractions from differentiation to proliferation and is a function of cell-secreted cytokines. (D) Proliferation rates of each cell type remain largely constant over the culture period. (E) death (or apoptosis) rates of all cell types are predicted to remain constant while there is a high level of SCF and nutrient (Glucose availability) and increases with nutrient depletion (F) SCF and Glucoase (GC) content is replenished every 2 days due to media exchange and, decreasing rapidly with increasing cell numbers.(A) The self-renewing fractions of LSK ((TIFF)Click here for additional data file.S4 Fig(A) The LT-HSC population declines over the entire culture period. (B) ST-HSCs recovers after day 4 of culture in a manner similar to the overall LSK cell growth observed in the 3-cell state model. (C) Similar kinetics are observed for MPPs and CMPs. (D) An exponential increase in the Terminal cell population is seen. Error bars represent standard error of mean.(TIF)Click here for additional data file.S5 Fig(A) self-renewing fractions of each sub-set rise steadily over time, also evidenced by the rising cell numbers. (B) fraction transitioning directly from ST-HSC or MPP to Terminal cells (jump). A higher number of ST-HSCs ‘jump’ to Terminal compared to MPPs, however each fraction is maintained over the entire culture period. (C) A higher fraction of LT-HSCs are predicted to remain quiescent while the more mature ST-HSC and MPP cells display a reduced quiescent capability. (D) The concentrations of differentiation stimulators and inhibitors in the culture media change dynamically over time, and is a function of cell-secreted cytokines. (E) Similar to the 3-state model, SCF and Glucose (GC) content is replenished every 2 days due to media exchange and, decreasing rapidly with increasing cell numbers. (F) Death (or apoptosis) rates of all cell types are predicted to remain constant while there is a high level of SCF and nutrient (Glucose availability) and increases with nutrient depletion.(TIFF)Click here for additional data file.S6 FigjSTHSCtoTerm and jMPPtoTerm values are set to 0, the model is unable to capture the experimental profiles for all cell populations. (A–E). The ST-HSC, MPP, and CMP populations exceed experimental observations, while Terminal cells are underpopulated due to lower initial differentiating cell numbers.When the (TIFF)Click here for additional data file.S7 FigRed nodes in the matrix indicate that model sensitivity is >1% for a 1% change in parameter values. This is indicative of high parameter sensitivity and system instability, but also design parameters for future experimental optimization. Similar to the 5-state model, Terminal cells, on account of their large and heterogeneous populations are relatively insensitive to several model parameters except their proliferation rates (PRTermmax).(TIFF)Click here for additional data file.S8 FigLSK, ProlifCMP etc.), others plateau or decrease over time. (D-F). These dynamic profiles are also observed for the 5-state model. Cell state response to these factors can help us identify the positive or negative impact of specific parameters over time, and whether culture modulation can further help regulate system response.(A-C) For the 3 state model, changes in sensitivity for select parameters for each cell type indicate that the system response is highly non-linear. While the impact of some parameters steadily rises over time (Apoptosis(TIFF)Click here for additional data file.S9 Fig(A) Schematic of the overall differentiation process with input and output flows associated with each cell type. The inputs correspond to increase in cell population  whereas outputs correspond to decrease in cell population . Rates associated with each flow are described in the equations given in the Supplemental section. (B) Schematic of the 3 exogenous soluble components of the system: Media, SCF, nutrient availability (denoted as GC for Glucose). Exchanging the media replenishes both components and is controlled by the parameter ‘change frequency’. (C) Concentrations of groups of biomolecules  are governed by the number of cells and a constant secretion rate associated with each cell type (c1 –c12) which dictate the self-renewing fractions  and the proliferation rates  for all cell types.(TIF)Click here for additional data file.S10 Fig(TIFF)Click here for additional data file.