To determine whether robot-assisted training is cost-effective compared with an enhanced upper limb therapy (EULT) programme or usual care.Economic evaluation within a randomised controlled trial.Four National Health Service (NHS) centres in the UK: Queen’s Hospital, Barking, Havering and Redbridge University Hospitals NHS Trust; Northwick Park Hospital, London Northwest Healthcare NHS Trust; Queen Elizabeth University Hospital, NHS Greater Glasgow and Clyde; and North Tyneside General Hospital, Northumbria Healthcare NHS Foundation Trust.770 participants aged 18 years or older with moderate or severe upper limb functional limitation from first-ever stroke.Participants randomised to one of three programmes provided over a 12-week period: robot-assisted training plus usual care; the EULT programme plus usual care or usual care.Mean healthcare resource use; costs to the NHS and personal social services in 2018 pounds; utility scores based on EQ-5D-5L responses and quality-adjusted life years (QALYs). Cost-effectiveness reported as incremental cost per QALY and cost-effectiveness acceptability curves.At 6 months, on average usual care was the least costly option (£3785) followed by EULT (£4451) with robot-assisted training being the most costly (£5387). The mean difference in total costs between the usual care and robot-assisted training groups (£1601) was statistically significant (p<0.001). Mean QALYs were highest for the EULT group (0.23) but no evidence of a difference (p=0.995) was observed between the robot-assisted training (0.21) and usual care groups (0.21). The incremental cost per QALY at 6 months for participants randomised to EULT compared with usual care was £74 100. Cost-effectiveness acceptability curves showed that robot-assisted training was unlikely to be cost-effective and that EULT had a 19% chance of being cost-effective at the £20 000 willingness to pay (WTP) threshold. Usual care was most likely to be cost-effective at all the WTP values considered in the analysis.The cost-effectiveness analysis suggested that neither robot-assisted training nor EULT, as delivered in this trial, were likely to be cost-effective at any of the cost per QALY thresholds considered.ISRCTN69371850. Our economic evaluation was designed and conducted following best practice methods which resulted in robust and generalisable results.Sensitivity analyses exploring any uncertainties surrounding the level of resource use and their impact on the cost-effectiveness of the interventions add to the robustness of the results.The unavailability of longer-term data for the within trial evaluation means that no robust inferences could be made on the long-term cost-effectiveness of the interventions.Poor completion of arm rehabilitation therapy logs meant that detailed information on the delivery of usual care was obtained from the health service utilisation questionnaire.The use of quality-adjusted life years based on responses to the EQ-5D-5L questionnaire as a generic outcome measure may not accurately capture changes in quality of life for this patient group.Stroke is the fourth leading cause of death in the UK and a leading cause of disability. Almost two-thirds of patients who had a stroke leave hospital with a disability.2 3The Robot-Assisted Training for the Upper Limb after Stroke (RATULS) trial sought to evaluate the clinical effectiveness and cost-effectiveness of robot-assisted training (using the MIT-Manus robotic gym system) by comparing it with either an enhanced upper limb therapy (EULT) programme, or usual care.5After conducting a scoping review, we found little evidence of cost-effectiveness studies in the UK. The only economic evaluation we found in the literature assessed the cost-effectiveness of robot-assisted training therapy for upper limb rehabilitation within the USA based VA Robotics study.The study was a three-arm RCT which recruited 770 participants from stroke units, day hospitals, outpatient clinics, primary care, community rehabilitation services and local stroke clubs in four study centres. The sample size calculation yielded a target sample size of 762 participants with 216 participants in each group required to provide 80% power at a significance level of 1.7%. The sample size was revised after protocol publication to 770 to allow for 15% attrition . Full details on sample size calculation and trial methodology have been reported elsewhere.4 5Participants were eligible for inclusion in the study if they were 18 years or older, had experienced a first-ever stroke between 1 week and 5 years prior to randomisation and, as a consequence of the stroke, had moderate or severe upper limb functional limitation as measured by the Action Research Arm Test (ARAT)The clinical primary outcome of the study was upper limb function ‘success’ using (ARAT)4 5We conducted an economic evaluation consisting of a cost–utility analysis using the quality-adjusted life year (QALY) as the primary outcome measure following guidance for best practice in health technology appraisal.11We conducted all analyses adopting the perspective of the UK National Health Service (NHS) and personal social services setting.The costs included in the analysis comprised the intervention costs for the robot-assisted training and EULT programmes, no intervention costs were directly associated with usual care and we assumed that any rehabilitation received had been reported in the health service utilisation questionnaire. We also included use of health and social services over the 6-month follow-up period for all randomised groups. For the intervention costs we assumed, as per the protocol,We developed a health service utilisation questionnaire, informed by previous data collection tools including the Client Service Receipt InventoryTo estimate the cost at 6 months for each participant we combined data on use of care and services with unit costs obtained from routine data sources. We applied NHS national reference costsWe used the information collected at 6-month follow-up in addition to the intervention costs to derive the total mean cost per participant per each randomised group. All costs are reported in pounds Sterling and converted to 2018 prices, when appropriate, using the Bank of England inflation calculator.18The quality of life outcome measures used for the economic evaluation were the summary utility scores derived from the EQ-5D-5L questionnaires.22For the main (base-case) cost-effectiveness analysis we only included those participants for whom we had some data on costs. Once we determined that missing cost data were missing at random, we explored in the sensitivity analyses the effect on cost-effectiveness of applying multiple imputation to missing total costs controlling for age, sex and baseline ARAT score.We explored the patterns of missing utility data. Once we established that information was missing at random, we used multiple imputation methods to estimate the missing utility values at 3 months and 6 months. This involved applying truncated normal regression while controlling for age, sex and baseline ARAT score in order to generate the missing utility value.We calculated mean costs and effects along with corresponding SD. Where we report differences in mean costs and effects between all three randomised groups we used 98.33% CIs, as this was a three-arm comparison. We conducted all pairwise comparisons using 95% CIs. Using seemingly unrelated regression modelling methodologyIn the analysis if a comparator was both more costly and less effective than the others it was dropped from any further cost-effectiveness comparisons because it was less cost-effective than the other comparator.We created cost-effectiveness acceptability curves in order to assess the imprecision surrounding the estimates of costs, effects and cost-effectiveness. This approach involved drawing bootstrapped samples, with replacement, of the mean costs and mean QALYs from the original trial data. We repeated this process increasing the number of replications until the results were stable. This was achieved at 1000 replications. After using the new values generated from the bootstrapping exercise to calculate the difference in costs and effects between groups, we combined this information with a range of willingness to pay (WTP) values  per QALY gained. This involved using the net benefit statistic26We conducted deterministic sensitivity analyses in order to assess the robustness of the cost-effectiveness results for three scenarios.First, we examined the impact of assigning a value of zero to missing total healthcare costs.Second, we examined the possibility that those participants with missing total healthcare costs may have used some services and hence incurred some costs. Under this scenario, once we established that information was missing at random, we applied truncated normal regression methods excluding total costs values below zero and above £25 000  and used age, sex and baseline ARAT as covariates.Finally, we investigated whether increasing the life span of the MIT-Manus robotic gym system from 5 to 7 years would affect the cost-effectiveness of the interventions.In order to explore the impact of time since stroke on the cost-effectiveness of the interventions, we conducted an exploratory analysis for the subgroups outlined in the protocol.A secondary per-protocol cost-effectiveness analysis removing from the data set those participants who did not receive at least 20 sessions of therapy in the robot-assisted training and the EULT programme groups was also conducted. The cut-off point of 20 sessions was based on clinical evidence that an additional 20 hours of therapy compared with control interventions leads to improvements on functional outcome.27We combined each sensitivity, subgroup and per-protocol analyses with bootstrapping in order to reflect the imprecision surrounding the cost-effectiveness results.We carried out a modelling exercise designed to extrapolate the mean QALYs at 6 months to 12 months based on the results of the trial, for this, we made a number of assumptions. First, we assumed that participants across all groups maintained the same utility levels reported at 6 months postrandomisation. Second, we considered all intervention costs and therapy-related costs  as sunk costs, since they were deemed not to continue beyond the 6-month trial period. However, we assumed that all other levels of healthcare resource use remained constant from 6 to 12 months postrandomisation.We calculated the difference in mean costs and effects between the randomised groups with differences across groups not being formally tested. We used seemingly unrelated regressionWe have designed and reported our research with input from stroke survivors. Members from the North East Stroke Research Network Patient and Carer Panel provided input to the design and content of trial documents, including health economics questionnaires.Most participants (96%) across all groups completed the health service utilisation questionnaire at baseline. There was a progressive increase in non-responses to the health service utilisation questionnaires over the 6-month follow-up period. The pattern of responses was similar across the intervention groups; however, the loss to follow-up was more pronounced in the usual care group. While completion rates were 83% for the robot-assisted training group and 85% for the EULT group, this decreased to 70% of usual care participants. Completion rate of EQ-5D-5L at baseline was 99% across all of participants. This decreased to 88% at 3 months and 82% at 6 months. The highest number of non-responders belonged to the usual care group with response rates of 81% at 3 months and 75% at 6 months.Reported health service resource use was broadly similar across all randomised groups at 6 months . Large SThe mean total cost per participant for each randomised group is reported in The mean utility scores across all randomised groups were similar at baseline, 3 months and 6 months as were mean QALYs . The EULThe base-case cost-effectiveness analysis results  show theThe results from the subgroup analysis are also summarised in Results from the per-protocol analysis did not change the direction of the cost-effectiveness results. Usual care remained the least costly option followed by EULT and robot-assisted training. The ICER for EULT and usual care was £68 000 and EULT only had a 17% probability of being cost effective at the £20 000 WTP threshold. The probability of robot-assisted therapy being considered cost-effective was very low.The different scenarios explored in these analyses did not change the direction of the results from the base-case cost-effectiveness results. Robot-assisted training remained dominated on average by EULT in all instances. First, when we changed the missing costs to zero, the resulting ICER between EULT and usual care increased to £172 000. This increase is to be expected since all participants with missing total costs belong to the usual care group. By imputing zero for missing costs the mean costs for the usual care participants decreased and hence, when this was done, the ICER increased when compared with EULT.Second, applying multiple imputation methods to missing costs resulted in an increase to the unadjusted mean usual care costs (£4451) compared with the base-case results (£3785). Consequently, the resulting ICER from the comparison between EULT and usual care decreased to £50 000 with the probability of EULT being cost-effective at £20 000 increasing to 27%.Third, extending the life of the robotic gym system resulted in a reduction of the mean capital costs per patient and hence, in a lower mean total cost for the robot-assisted training group (£5085) compared with the base-case analysis (£5387). There were no changes to the utility scores across all groups nor to the mean costs for EULT and usual care, hence the resulting ICER for the comparison of EULT and usual care remained the same as in the base-case analysis (£74 100).The RATULS trial found no evidence that robot-assisted training, as delivered in the study, improved upper limb function ‘success’ for patients who had a stroke with moderate or severe upper limb functional limitation when compared with a EULT programme or usual care.Results from the base-case cost-effectiveness analysis suggested that, on average, robot-assisted training was more costly than both EULT and usual care and that robot-assisted training was slightly less effective than EULT. EULT was on average more costly and as effective as usual care in the unadjusted analyses and more costly and more effective in the adjusted analyses. The balance of probabilities favoured usual care as the preferred upper limb rehabilitation therapy over the range of WTP values considered. Focusing on the society’s WTP for a QALY, the bootstrapped analysis suggested that, EULT, as delivered in this trial, was unlikely to be cost-effective over any of the WTP values considered, despite being more effective than robot-assisted training and usual care. The subgroup, sensitivity and per-protocol analyses did not change the direction of the base-case cost-effectiveness results. Extrapolating within-trial results to 12 months produced the lowest ICER for the comparison between EULT and usual care overall, however, high uncertainty surrounds the assumptions made about how costs and utilities change beyond the trial follow-up.The main strength of this economic evaluation is that it was conducted as part of a rigorously run RCT and followed guidelines for best practice throughout.One of the main challenges in conducting the economic evaluation was the difficulty to ascertain the specific components of usual care therapy. Log books designed to capture detailed usual care information were completed by participants. The information gathered was to be used alongside the health service utilisation questionnaire. Completion rates, however, were very low and we were unable to incorporate these data into the economic evaluation. We overcame this by drawing on the information captured via our primary data collection tool, the healthcare service utilisation questionnaire, where participants recorded any therapy sessions received during the trial period.Through the collection of self-reported quality of life information at three points during the study using the EQ-5D-5L questionnaire,A noteworthy limitation of the economic evaluation is associated with the timeframe of the trial. The within-trial economic evaluation assessed the cost-effectiveness of the interventions at 6 months. A longer-term perspective was originally planned but due to limitations of the data, extrapolation to 12 months only was conducted. The results however, need to be interpreted with caution due to the assumptions made on both costs and utility values.The economic evaluation fills a significant evidence gap with this being the first economic evaluation comparing robot-assisted training with usual care having been conducted in the UK NHS setting. This evaluation expands the analysis conducted on the cost-effectiveness of the MIT-Manus robotic system as part of the VA Robotics trial.The use of multiple sites contributed to the generalisability of the economic evaluation. The analyses controlled for differences in sites hence minimising the chance of obtaining biased results from differences in costs and effects driven by location.In conclusion, robot-assisted training was not found to be cost-effective in comparison to EULT and usual care. This economic evaluation suggested that usual care remained the most cost-effective type of upper limb rehabilitation compared with a EULT therapy programme and robot-assisted training for patients who had a stroke with moderate or severe functional limitation.The results create opportunities for further research. In particular, further research could explore the potential effect on both costs and QALYs from reconfigurations to the delivery of EULT and robot-assisted training. It remains unclear, for example, whether delivering therapy in a group setting may first, be feasible in the NHS setting and second improve the quality of life and clinical outcomes. Further development of these interventions may increase their cost-effectiveness compared with usual care. Additionally, studies with a longer follow-up data may help establish whether the QALY gains derived from the interventions are sustained beyond the set timeframe of the trial, this can then lead to a robust assessment of their long-term cost-effectiveness.


Further screening had suggested that repositioning of the S-ICD lead inferiorly would result in acceptable sensing; however, when system revision was planned to be undertaken the patient failed repeat S-ICD screening, with no appropriate vector identified for the current Micra AV / WiSE-CRT combination. It was therefore decided to implant a further Micra AV device in a superior position in the septum / RV outflow tract. Given the patient’s poor LV systolic function and comorbidities, the prior Micra devices were not extracted. In this location there were acceptable sensing (4.3 mV) and pacing (0.5 V at 0.4 ms) parameters and a stable “tug test.” S-ICD sensing with pacing from the new Micra AV location and the WiSE-CRT system was satisfactory F and theThis case report demonstrates an important learning point for the management of patients with multiple leadless cardiac devices. While there was appropriate sensing by the S-ICD after initial implant of a Micra leadless pacemaker and WiSE-CRT system, implantation of a new Micra in a different location within the RV changed the biventricular paced QRS morphology, resulting in suboptimal S-ICD sensing. On 2 sensing vectors, T-wave oversensing was noted, which carries a risk of inappropriate shocks. On the remaining sensing vector the biventricular paced QRS complex was low in amplitude, which required an increase in the device sensitivity and resulted in oversensing of myopotentials and an inappropriate shock. Implantation of a third Micra resulted in a significantly different RV and biventricular paced ECG, which resulted in satisfactory S-ICD sensing.Inappropriate sensing by S-ICD devices can be problematic, with 7%–15% of patients failing initial screening,Our case demonstrates that particular care should be taken when a replacement leadless pacemaker is implanted if in combination with an S-ICD, as the resulting change in morphology of the paced QRS complex may cause suboptimal S-ICD sensing. Intraprocedural S-ICD screening is therefore recommended during pacing from the new leadless pacemaker position to ensure adequate sensing prior to deployment. Patients may require the implantation of multiple leadless pacemakers in their lifetime, owing to battery depletion or device failure. The estimated battery longevity of the Micra leadless pacemaker is 12 years•A completely leadless cardiac resynchronization and defibrillator system can be achieved using a Micra leadless pacemaker, WiSE-CRT system, and a subcutaneous implantable cardioverter-defibrillator (S-ICD).•In patients with both a Micra leadless pacemaker and an S-ICD, implantation of a subsequent Micra may change the paced QRS morphology and affect S-ICD sensing, thus highlighting the importance of Micra position within the right ventricular cavity.•Intraprocedural S-ICD screening to determine adequate sensing from the new Micra position is recommended prior to deployment.•In such patients, extraction of the existing Micra may be preferable when a replacement device is required, to allow implantation in the same position as the original device, though this may be associated with risks, particularly in patients with left ventricular impairment.This case highlights the importance of the position of the leadless pacemaker within the RV cavity, as differing paced QRS morphologies may affect S-ICD sensing. This may be an increasingly common and important issue in the future with the increased use of leadless technologies.


Nonlinear phenomena are universal in ecology. However, their inference and prediction are generally difficult because of autocorrelation and outliers. A traditional least squares method for parameter estimation is capable of improving short-term prediction by estimating autocorrelation, whereas it has weakness to outliers and consequently worse long-term prediction. In contrast, a traditional robust regression approach, such as the least absolute deviations method, alleviates the influence of outliers and has potentially better long-term prediction, whereas it makes accurately estimating autocorrelation difficult and possibly leads to worse short-term prediction. We propose a new robust regression approach that estimates autocorrelation accurately and reduces the influence of outliers. We then compare the new method with the conventional least squares and least absolute deviations methods by using simulated data and real ecological data. Simulations and analysis of real data demonstrate that the new method generally has better long-term and short-term prediction ability for nonlinear estimation problems using spawner–recruitment data. The new method provides nearly unbiased autocorrelation even for highly contaminated simulated data with extreme outliers, whereas other methods fail to estimate autocorrelation accurately. A nonlinear curve is frequently used to model SR relationships. However, it is notoriously difficult to estimate SR relationships accurately, especially for many fish stocks5, given that SR data often have large deviations around the SR curve, including extreme outliers, and residuals left over after SR curve fitting are likely to show strong autocorrelation. These outliers and autocorrelations make statistical inference difficult and rarely permit accurate estimation of existing SR relationships hidden in the data of a single population.Nonlinear modeling is widely applied to ecological data analysis. The spawner-recruitment (SR) relationship, which is also called the stock-recruitment relationship, is fundamental to population dynamics modeling, risk assessments in conservation biology, and sustainable use of wildlife6 is an influential method for overcoming the difficulty of finding evidence of density dependence from individual population data. In contrast with individual analysis, meta-analysis of SR data elicits firm conclusions and facilitates evidence-based decision making by integrating the parameters and/or results obtained by fitting a nonlinear model to time series data on abundance and SR relationships8. For instance, Brook & Bradshaw (2006) fitted nonlinear models to the abundance time series data of 1198 species and integrated the results to detect evidence for density dependence9. Although they found that density dependence is a pervasive feature of population dynamics in various species, support for the density dependence of fish was generally weaker than those of other taxa . This lack of power to detect density dependence could be caused by excessive noise , outliers, and the complex correlation structure involved in individual SR relationships in fish. The improvement in quality of the result from an individual dataset using a robust estimation method against noisy data is therefore of crucial importance even in meta-analysis.Meta-analysis of information from multiple independent studies10 cannot handle autocorrelated residuals, and subsequent research demonstrated that the method was highly sensitive to small perturbations in the SR data11. A regression model that is insensitive to perturbations that deviate from the mean relationship and is sensitive to the autocorrelation of residuals must mitigate the trade-offs between long-term and short-term objectives for sustainable development.Predictive ability based on SR relationships is closely related to sustainable fisheries management. Setting proper long-term management objectives is necessary for sustainable use of fish, whereas fishers and fish processors are usually concerned with short-term prediction of catches for logistics and planning for the next season. Balancing the trade-offs between long-term and short-term management objectives will therefore enhance compliance with fisheries regulations, thereby avoiding overfishing and consequently realizing sustainability goals. The credibility and benefits of long-term management objectives depend on the accuracy and precision of the parameter estimates of SR relationships, which provide biological reference points based on maximum sustainable yield. In contrast, short-term predictive ability is strongly influenced by the magnitude and autocorrelation of residuals as well as parameter estimates of the SR relationship. Achieving accurate and precise long-term and short-term prediction from noisy SR data contributes substantially to the sustainable use of fish. A robust method previously developed for estimating fish recruitment12.In this work, we propose a new robust regression approach by extending a least squares (LS) method to realize a weighted maximum likelihood method with changeable variance and autocorrelation dependent on deviance residuals. The new approach accurately estimates parameters and precisely predicts autocorrelated error structures, even for contaminated SR relationship data containing many outliers. The approach is tested using simulated SR data having some outliers and autocorrelated error structures. The results are compared with those from traditional regression models that use an LS method and a least absolute deviations (LAD) method. Further, we apply our robust regression approach to compiled fish spawner-recruitment data from JapanS) and recruitment (R) observed over time. A lognormal distribution is frequently used as the distribution of errors for SR relationships13. We therefore assume that the residuals from a regression model having 5.The data needed for estimating the SR relationship consist of spawning biomass  ppendix A,σ2/λt ppBy solving the equation at equilibrium, the mean deviance residual at 15, which is also called retrospective forecasting16 (RF), to stably determine the value of P17.We need to estimate the parameters 18 and its package TMB19 (Template Model Builder).All subsequent analyses are performed using R11. The average recruitment data are generated from the Hockey–Stick (HS) SR function12, q). We then have eight types of simulated data  is set to 30 years which is typical for SR time series data12. The performance of the methods is evaluated by two indicators that represent long-term and short-term predictive abilities 20. The parameter We generate the simulated data  same as the above base case scenario (S0) except that 5. The mathematical details are given in Appendix Finally, we calculate biological reference points related to maximum sustainable yield (MSY), i.e., fishing rate at MSY  fitted the SR curves to fish population data from Japan which comprise 26 SR datasets Appendix , demonstWhen the simulated data are generated without autocorrelation and outliers in the residuals, LS performs best because the true and estimation models are then entirely in agreement, and LAD and RSR also produce nearly unbiased results with only slightly worse precision Fig. . When thWhen the simulated data are generated with autocorrelated residuals and no outliers/two-sided outliers, LS, LAD, and RSR produce nearly unbiased estimates with similar accuracy and precision Fig. . LAD prob is estimated with almost no bias for all estimation methods and all scenarios, even though the precision of the LAD method is inferior to other methods  in the Sea of Japan shows a linear relationship (no density-dependence), the RSR-based SR curve shows a break point within the observed spawner abundances  in the Tsushima warm current shows a flat relationship (extremely strong density-dependence), the RSR-based SR curve also shows a break point within the observed spawner abundances  using 22. Although we used a normal distribution in this case, we can use other probabilistic distributions in our modeling framework. The time series cross-validation or retrospective forecasting for selecting the optimal 14 will be topics of future research.The approach used in this paper is applicable to various robust regression problems, not to mention a linear regression with outliers. Although we dealt with just a one-year time lag or autoregressive process of order 1 in this paper, the RSR method is easily extended to an autoregressive process with higher orders AR is frequently used to model nonlinear population dynamicsers Fig.  and, as 28. Ecological data are often simultaneously contaminated by inevitable but obstructive outliers and influenced by autocorrelative phenomena. The outliers make long-term and short-term prediction difficult, whereas autocorrelation affects the long-term and short-term prediction and may even distort the estimation results of the latent nonlinear structure. Traditional robust regression approaches alleviate the influence of outliers but make estimation of autocorrelation difficult. Our RSR reduces the influence of outliers and accurately estimates the innate autocorrelation, thereby greatly improving long-term and short-term prediction ability compared with traditional robust regression approaches. Accordingly, RSR holds promise for extensive applications and may prove useful for various ecological problems.Robust nonlinear regression analysis is potentially applicable to extensive ecological time series data, including not only SR data as in the present work, but also radioisotope contamination dataSupplementary Information.


Peripheral blood transcriptome is a highly promising area for biomarker development. However, transcript abundances (TA) in these cell mixture samples are confounded by proportions of the component leukocyte subpopulations. This poses a challenge to clinical applications, as the cell of origin of any change in TA is not known without prior cell separation procedure. We developed a framework to develop a cell-type informative TA biomarkers which enable determination of TA of a single cell-type (B lymphocytes) directly in cell mixture samples of peripheral blood  without the need for subpopulation separation. It is applicable to a panel of genes called B cell informative genes. Then a ratio of two B cell informative genes (a target gene and a stably expressed reference gene) obtained in PBMC was used as a new biomarker to represent the target gene expression in purified B lymphocytes. This approach, which eliminates the tedious procedure of cell separation and directly determines TA of a leukocyte subpopulation in peripheral blood samples, is called the Direct LS-TA method. This method is applied to gene expression datasets collected in influenza vaccination trials as early predictive biomarkers of seroconversion. By using TNFRSF17 or TXNDC5 as the target genes and TNFRSF13C or FCRLA as the reference genes, the Direct LS-TA B cell biomarkers were determined directly in the PBMC transcriptome data and were highly correlated with TA of the corresponding target genes in purified B lymphocytes. Vaccination responders had almost a 2-fold higher Direct LS-TA biomarker level of TNFRSF17  on day 7 after vaccination. The sensitivity of these Direct LS-TA biomarkers in the prediction of seroconversion was greater than 0.7 and area-under curves (AUC) were over 0.8 in many datasets. In this paper, we report a straightforward approach to directly estimate B lymphocyte gene expression in PBMC, which could be used in a routine clinical setting. Moreover, the method enables the practice of precision medicine in the prediction of vaccination response. More importantly, seroconversion could now be predicted as early as day 7. As the acquired immunology pathway is common to vaccination against influenza and COVID-19, these biomarkers could also be useful to predict seroconversion for the new COVID-19 vaccines. Vaccination by controlled exposure to an antigen or its precursor is a good strategy for prevention of full-blown infection. Such prior exposure activates the acquired immune system to produce antibodies against the pathogen. In vaccinated individuals, the pathogens will be controlled quickly, and symptoms of infection are alleviated. A common example is vaccination against the influenza virus. As the prevalent strains of influenza virus change frequently, annual vaccination of different influenza virus strains is a common practice. Of particular relevance is the current pandemic COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), against which a worldwide vaccination campaign is also underway.In the case of influenza vaccination, only about half of those vaccinated individuals mount substantial immune responses against the antigens, which is known as seroconversion. They are called responders (R) to vaccination and the remaining responses are called non-responders (NR). However, antibody production, measured in the unit of titers representing the level of antibodies against the vaccination antigen in blood samples, can only be known 28 days after vaccination . In ordeBlood transcriptome biomarkers is a subject undergoing intensive research ,3,4. In Most vaccination transcriptome studies used PBMC samples, while only a few studies used WB samples . These tTo improve our understanding of the cellular origin of a particular gene transcript commonly used in peripheral blood mixture samples, a workflow flowchart was derived to define a list of cell-type informative genes in a peripheral blood mixture sample . In esseA mathematically based methodology (known as deconvolution) has been used to determine the proportional cell counts of various subpopulations in cell mixtures, which requires a long list of signature genes for each subpopulation ,15,16. HWith the list of B lymphocyte target genes identified with our model, we could estimate their expression in the B lymphocytes directly in peripheral blood. We analyzed these B lymphocyte TA biomarkers in publicly available datasets of vaccination trials. As we were interested in identifying early B lymphocyte TA biomarkers that can predict subsequent seroconversion, we explored the predictive performance of the new biomarkers.In order to identify B lymphocyte cell-type informative genes that can predict vaccination response, the following datasets of gene expression obtained from peripheral blood samples were used . These dResponders (R) after vaccination were defined following the criteria of a seroconversion/significant increase of anti-hemagglutinin antibody levels (HI titer) performed by hemagglutination inhibition (HI) assays on subjects’ plasma or serum which was taken before and after vaccination (commonly taken on Day 28) . The EurAn overview of the workflow to define B lymphocyte informative genes and their application in the meta-analysis is shown in a flowchart in the To identify B lymphocyte informative genes among over 10,000 genes that are expressed in hematological cells for downstream analysis, a mathematic model was used to shortlist putative genes by incorporating an expected proportional cell count of a particular cell-type (B-lymphocyte in this study) in a cell-mixture sample . Although the B lymphocyte count is variable among individuals and decreases with age ,21, a baThe model is explained in more detail in GAPDH, UBC), one (or more) reference gene was chosen among the informative genes with an additional requirement of having a low biological variation. This was because conventional housekeeping genes are produced by all the different cell-types in the cell mixture, so it only represents the total cell count of all those various cell-types present in the cell mixture sample but does not represent the cell count of B lymphocytes. Therefore, the normalization factor also must have been a cell-type informative gene. The biological variation could be calculated as inter-individual variance in the purified cell-type samples and expressed as a coefficient of variation (CV%).As transcripts of these informative genes predominantly come from a single cell-type, direct measurement of their total TA in the cell mixture sample would provide a valid estimation of the gene expression level of that component cell-type. However, we needed a reference gene for normalization for the biological variation in the proportional cell counts among individual samples. Instead of using traditional housekeeping genes  and its ground-truth, TA of that target gene in purified B lymphocytes (TA target gene/TA standard housekeeping gene in purified B lymphocyte samples). The Direct LS-TA method can be applied to more than 10 target genes and B lymphocyte expression of these genes could be reliably determined directly in PBMC.A mathematical model to identify subpopulation informative genes was derived to determine the required fold difference threshold between a purified cell sample and a cell mixture sample for any given proportional cell count. The threshold value facilitates the identification of cell-type informative genes for various scenarios of different cell-types in different cell-mixture samples  .The following model can be used to determine the relationship between % contribution by a cell type based on the differential gene expression of purified cell samples and a cell-mixture sample.Let= total transcript abundance (TA) of a gene in a WB sample= TA of a gene in B cells present inside the WB sampleThis value is assigned based on general knowledge or (in other settings) could be estimated from sample data by methods like deconvolution using TA data of other genes.= proportional cell count of B cells inside the WB sample = transcript abundance of all other cell-types based on their respective proportional counts. X= This is the observed difference in expression of a gene purified B cell samples and WB.= fold change difference between Y-axis in X-axis).In order to understand the relationship between % contribution of TA in a cell mixture (WB) by a specified cell-type  between specified cell type and WB is linear with the slope defined by the proportional cell count of that cell type.The expression indicates that the relationship between % A RNA-seq dataset (GSE45764) had transcriptome data of both purified B lymphocytes and PBMC. Therefore, the datasets were used to identify B lymphocyte informative genes. Next, biological variation was determined for all B lymphocyte informative genes and expressed in terms of CV%. TNFRSF13C and FCRLA were selected as a B lymphocyte reference genes, respectively, which were used as the denominator in the new Direct LS-TA biomarker parameter to infer B lymphocyte expression of the target genes.As log transformation had been applied to the TA data, this new biomarker parameter was calculated as the difference (subtraction) between the B lymphocyte informative target gene and the B lymphocyte informative reference gene. Using TNFRSF17 as the target gene, for example, its Direct LS-TA could be expressed as:As the dataset GSE45764 provided RNAseq transcriptome data of paired PBMC and purified B lymphocytes samples taken at the same time. The ability of the Direct B lymphocyte LS-TA resulted in reflecting the ground-truth, while TA of the same target gene in purified B was evaluated by the correlation between the Direct B lymphocyte LS-TA in the PBMC samples and the TA of the target genes in the purified B lymphocytes. Pearson’s correlation coefficient (r) was used. A conventional housekeeping gene  was used to normalize the TA results in the purified B cell samples .Using this exploratory dataset, the Pearson’s correlation coefficient (r) values of all shortlisted B lymphocyte informative genes were determined, and only those with r > 0.8 were further analyzed for their differential expression between R and NR groups.In addition, several other datasets  also contained microarray expression data of both PMBC and purified lymphocytes samples. They were also used to validate the list of B lymphocyte informative genes, reference genes and the performance of Direct B lymphocyte LS-TA of the selected target genes.To have a better idea of the reproducibility of these findings, meta-analyses were performed for these four Direct B lymphocyte LS-TA biomarkers with PBMC data in a collection of datasets that were performed on different platforms (including Affymetrix and Illumina microarrays).The datasets were downloaded from GEO. Direct LS-TA results were determined from expression intensities of the target genes and reference genes that had been picked up in the discovery dataset. Fixed effect meta-analysis was performed using R package meta.The Microarray datasets were checked to see whether they had been normalized by RMA normalization or quantile normalization. All data were also log-transformed with base 2. The quality check of the datasets included a check for outliners by Mahalanobis distance metrics ,24 usingStatistics analyses were performed with R packages, including plotROC, meta and stats.A list of B lymphocyte informative genes is shown in The correlation between the new Direct LS-TA biomarkers in PBMC samples and the ground-truth TA in purified B lymphocytes was used to assess its ability to infer expression of the target genes in purified B lymphocytes.Y-axis with the expression level of that gene in paired purified B lymphocytes shown on the X-axis. The strong correlation  results confirmed that Direct LS-TA TNFRSF17 in PBMC is a reliable indicator of TNFRSF17 expression in B lymphocytes.Similarly, Direct LS-TA for another target gene (TXNDC5) in PBMC was also representative of its expression in B lymphocytes. This was shown in both RNAseq data and microarray validation data. With such a strong correlation, Direct LS-TA quantification was applied to other vaccination response datasets in which only PBMC samples were collected. In fact, the great majority of vaccination response datasets did not have expression information for purified leukocyte subpopulations. The feasibility of using the Direct LS-TA method provides a means to analyze cell-type specific TA in these and many other cells-mixture datasets.Not only could the B-lymphocyte expression of TNFRSF17 be inferred directly in a peripheral blood sample, but a list of other B-lymphocyte informative genes (IGHG1 and IGLL5). Other target genes include MZB1, FCRL5, TNFRSF17 and TXNDC5, which have been associated with various essential functions of B lymphocytes.Many target genes encode important proteins for antibody production .With this new B lymphocyte biomarker available , it is nSimilarly, Direct LS-TA of another target gene TXNDC5 (both Direct B lymphocyte LS-TA of TXNDC5 on Day 7 and the increments from Day 0 to Day 7) were also significantly different between responders and non-responders .To have a better idea of the reproducibility or replication of these findings, meta-analyses were performed for these four Direct B lymphocyte LS-TA biomarkers in PBMC data in a collection of datasets. These data were obtained from different transcriptome quantification platforms (Affymetrix and Illumina microarrays).The meta-analysis on TNFRSF17 was performed on 7 PBMC datasets . RegardlSimilarly, the increment from Day 0 to Day 7 was analyzed by meta-analysis in these datasets. The overall effects of greater increment in the responders were confirmed .Receiver operator curve (ROC) analysis was used to evaluate the test performance of Direct LS-TA as an early (first-week) predictive biomarker for subsequent seroconversion on Day 28. As the case-mixes were different among studies, the Area Under Curve (AUC) values were also different . All stuThe exact cut-off point to be used depends on the purpose and rationale of the test. The applications of this method are wide. For example, if the vaccine is unstable, the producer would want to know early if any batch of the vaccine had degraded; this test would provide the answer as early as seven days after administration of the batch. In this scenario, a group of vaccine recipients will be monitored for the Day-7-increase in Direct B lymphocyte LS-TA. A high sensitivity cut-off value would be used, so most of the recipients are expected to reach the cut-off value indicating that the vaccine is active. If the number or percentage of recipients showing an increase in Direct B lymphocyte LS-TA on Day 7 is less than a pre-defined percentage, degradation of the vaccine batch would be possible and remedial work could be implemented early rather than waiting for the seroconversion data after Day 28.In addition, the Direct B-lymphocyte LS-TA test could be used for personalized vaccination in which an individual wants to know his subsequent vaccination response early after vaccination. A high specificity cut-off value would be used in this scenario. Although only around 60% of the responder recipients would achieve this cut-off value, the false position rate is low. This supports a high positive predictive value, so recipients with positive Direct B-lymphocyte LS-TA results would be very likely to be responders. The consequence of a negative Direct B-lymphocyte LS-TA is a requirement of doing a follow-up antibody test on Day 28. The high positive rate of Day 7 Direct B lymphocyte LS-TA allows the majority of responders to obtain their vaccine response results early.The Direct LS-TA biomarker is a simple B-lymphocyte informative TA biomarker that allows direct determination of B lymphocyte gene expression in PBMC samples without the need to purify B lymphocytes from other blood cells.This approach is different from deconvolution methods of deriving the proportional cell counts of various cell-type subpopulations . On the Previous studies including those datasets we analyzed here provided comprehensive and long lists of differential expression genes (DEGs) typically composed of tens or hundreds of genes . Then, pOn the other hand, the new biomarker using a ratio of TA of only two cell-type informative genes is both technically straightforward and easy to interpret. Here, the key hurdle is to identify these cell-type informative genes for which their TA could be readily estimated in cell mixture samples. In this paper, we showed a panel of genes that could be used for this purpose and their application in a real life situation.https://www.proteinatlas.org/search/blood_cell_lineage_category_rna%3Ab-cells%3BLineage+enriched+AND+tissue_category_rna%3ABlood%2CLymphoid+tissue%3BIs+highest+expressed+AND+sort_by%3Atissue+specific+score (accessed on 4 May 2021)). Here, the two terms, B lymphocytes informative genes and B lymphocyte lineage enriched genes are conceptually related but the gene lists are slightly different, as the methods of calculation are different. For example, key target genes  used as the biomarker in this paper are not included in the protein atlas cell-type enriched gene list of B lymphocytes. Here, we chose two reference genes (TNFRSF13C and FCRLA), as they had low CV% similar to other known B lymphocyte-specific genes . Other reference genes could also be used but prior validation was required.Based on a similar concept as our cell-type informative genes, the latest human protein atlas  also hasOur analysis was limited by (1) availability of study datasets and (2) having little control of the study protocol of the original study. For example, there were studies using different gene expression quantification platforms, ranging from microarrays to RNA-sequencing. Anyway, the versatility of our methods enable comparison and meta-analysis of data generated from different platforms.Another limitation was due to the same sample size in each dataset. Four datasets had less than 50 participants. Furthermore, the response rate was heavily tilted toward either side in several studies with either a very high or very low response rate. Therefore, meta-analysis was only applied to those datasets having a response rate between 10–90%.A new and simple to analyze peripheral blood biomarker is introduced here which can be readily incorporated into the routine clinical laboratory. It could predict seroconversion status early after influenza vaccination. A global-wide vaccination campaign is underway to control the COVID-19 pandemic. Due to using the same mechanism of antibody production, Direct B lymphocyte LS-TA will also be a useful companion assay for the practice of personalized vaccination.


Macular coloboma is a rare eye condition that affects around 0.5-0.7/10,000 of live births. Macular coloboma appears as a well-demarcated atrophic lesions that could affect one eye or both eyes on fundus examination. This is a case of a 33-year-old male patient who presented to the outpatient clinic with a history of poor vision in the left eye since childhood. He had a history of strabismus surgery for sensory exotropia (XT) in the left eye. Anterior segment examination of both eyes was normal while the fundus examination of both eyes revealed bilateral chorioretinal lesions in the macula which was larger in the left eye (OS) than the right eye (OD), representing bilateral chorioretinal coloboma. Congenital coloboma is a rare eye condition that leads to non-progressive decrease in visual acuity. Optical coherence tomography (OCT) is the modality of choice in diagnosing and describing macular coloboma. Coloboma is an ocular congenital defect that can affect different areas of the eye such as the iris, the retina, the choroid, the lens or the optic nerve . MeanwhiA 33-year-old male patient presented to our outpatient clinic with a history of poor vision in the left eye since childhood. On eliciting the past ocular history, he had left eye strabismus surgery six years ago to correct sensory exotropia (XT). Previous medical history was positive for type 1 diabetes mellitus, and is controlled by insulin injections. His family history of ocular disease and systemic disease was unremarkable. Ophthalmic examination showed that the best-corrected visual acuity of the right eye (OD) was 20/30, and the left eye (OS) was hand motion. Intraocular pressure (IOP) on the non-contact tonometer was 16 mmHg and 12 mmHg in the right and left eye, respectively. The anterior segment examination was unremarkable in both eyes. Eye alignment by Hirschberg test and cover-uncover test showed orthotropic alignment in both eyes. Extraocular muscle movement showed a full range of movement without limitation bilaterally. The fundus examination (OD) showed a small circular well-circumscribed pigmented chorioretinal atrophic lesion. It had a well and sharp demarcated borders measuring around a half-disc diameter in both horizontal and vertical dimensions without involving the center of the fovea (Figure Macular coloboma results from incomplete or poor differentiation of arcuate bundles along the horizontal raphe, which normally occurs during fetal development ,5. On thAbsence of the retina and choroid layers was reported using OCT in our patient. These findings are comparable to what has been reported in the literature ,7-10. FuMacular coloboma, especially those located at the posterior pole, can lead to retinal detachment leading to an increase risk of developing complete vision loss . TherefoCongenital coloboma is a rare ocular condition that leads to non-progressive decrease in visual acuity and if not followed up regularly may cause retinal detachment and vision loss. Optical coherence tomography is the method of choice in diagnosing macular coloboma while treatment should be done by laser photocoagulation if the coloboma is away from the macula with careful observation and follow-ups.


In the current work, a pix2pix conditional generative adversarial network has been evaluated as a potential solution for generating adequately accurate synthesized morphological X-ray images by translating standard photographic images of mice. Such an approach will benefit 2D functional molecular imaging techniques, such as planar radioisotope and/or fluorescence/bioluminescence imaging, by providing high-resolution information for anatomical mapping, but not for diagnosis, using conventional photographic sensors. Planar functional imaging offers an efficient alternative to biodistribution ex vivo studies and/or 3D high-end molecular imaging systems since it can be effectively used to track new tracers and study the accumulation from zero point in time post-injection. The superimposition of functional information with an artificially produced X-ray image may enhance overall image information in such systems without added complexity and cost. The network has been trained in 700 input (photography)/ground truth (X-ray) paired mouse images and evaluated using a test dataset composed of 80 photographic images and 80 ground truth X-ray images. Performance metrics such as peak signal-to-noise ratio (PSNR), structural similarity index measure (SSIM) and Fréchet inception distance (FID) were used to quantitatively evaluate the proposed approach in the acquired dataset. Drug discovery aims in identifying novel candidate treatments against diseases. It is a lengthy (it takes about 15 years) and costly (about 1–1.5 billion euros) process involving a laborious series of different stages, including preclinical studies .A crucial part in the preclinical evaluation of drug discovery is performed on the basis of biodistribution ex vivo studies that aim in monitoring the interaction of radioisotope- or fluorescence-labeled candidate drugs on animal subjects invasively. However, this invasive method, although a gold standard, raises economical and ethical issues due to the large number of required animals . Non-invPlanar molecular imaging is another alternative since it can effectively be used to track a new tracer and study the accumulation from zero point in time post-injection. Two-dimensional fluorescence/bioluminescence optical imaging is a well-established method for this purpose ,8, offerThe aim of this work is to explore a potential solution for generating adequately accurate synthetic morphological X-ray images by translating standard photographic images of mice using deep learning. Such an approach would benefit planar molecular imaging studies since the acquired photographic images have poor resolution in terms of anatomical information. By superimposing adequately high-resolution synthesized anatomical images on real functional mouse images, it would be possible to generate a preliminary estimation of the drug distribution within the body of the animal using conventional photographic sensors, thus reducing cost and complexity by eliminating the need for X-ray imaging of the animal.For this purpose, we trained and tested a well-known conditional generative adversarial network for image-to-image translation (pix2pix cGAN) ,13 in a A pix2pix cGAN was trained to map conventional photographic mouse images to X-ray scans . Pix2pixA dataset of 780 input/ground truth images has been acquired in order to train and test the pix2pix network. A photographic mouse image and the corresponding X-ray scan of the same anesthetized animal are referred to as input/ground truth images, respectively. The input images have been acquired using two commercial planar scanners for SPECT and PET radioisotope imaging, which contain a standard photographic sensor that provides an optical image of the animal as an anatomical mapping solution  a. The X-We acquired 5 input/ground truth images of each animal in different poses upon the hosting bed. Given that small mice used in preclinical studies have similar dimensions and weight (∼20–40 g), we assume that the aforementioned methodology does not affect the training procedure and minimizes the number of laboratory animals used in the study. In all cases, animals were anesthetized with isoflurane and kept warmed during the scans. All animal procedures were approved by the General Directorate of Veterinary Services  and the Bioethical Committee of the Institution (Permit number: EL 25 BIOexp 04).The study involved 78 white and 78 black Swiss albino mice, leading to a total number of 780 input/ground truth images. The mice were classified into two groups: (i) a group of 70 white and 70 black mice in order to collect the 700 paired images for training; (ii) a group of 8 white and 8 black mice in order to collect the 80 paired images for test and validation. Except mouse color, the method was evaluated against different animal hosting beds , which leads to different backgrounds in the input image. Four indicative input/ground truth pairs used for training and/or testing are illustrated in y from a set of known images x and a randomly created noise vector z as follows:Pix2pix is a cGAN  deep learning network that can be used for image-to-image translation. A cGAN is designed to create synthetic images G) and the discriminator (D). The generator  peak signal-to-noise ratio (PSNR), (b) structural similarity index measure (SSIM) and (c) Fréchet inception distance (FID). PSNR is an expression for the ratio between the maximum possible signal power value and the power of distorting noise that affects the quality of its representation ; SSIM coQuantitative evaluation of network outputs compared to ground truth images has been performed using three performance metrics: (a) PSNR; (b) SSIM; (c) FID in the test dataset. According to the calculated values presented in The proposed trained network was used for anatomical mouse mapping in two, proof of concept, nuclear molecular imaging experiments. Two planar preclinical scanners for SPECT and PET isotopes imaging have been used . The scanners come with a pre-installed photographic sensor, which is used to superimpose standard photography of the mouse with the acquired nuclear image for anatomical mapping purposes. The trained with the Cross Entropy loss function pix2pix network was used to generate an X-ray scan from the acquired photographic image in real-time during in vivo experiments. The output was then superimposed in real-time with the corresponding functional (PET or SPECT) image to provide high-resolution information for anatomical mapping.Two healthy mice were administered through tail vein injection with 286 uCi/50 uL of technetium-99m  that learns a mapping from the input image to the output image has been trained in a 700 input (mouse photography)/ground truth (mouse X-ray scan) dataset and evaluated in a 80 input/ground truth dataset. The pix2pix network has been evaluated against three different loss functions: (a) Cross Entropy, (b) MSE and (c) Wasserstein distance, with the first two achieving similar performance superior to the Wasserstein distance. However, the results in all cases show that the network predicts an X-ray image with sufficient accuracy and that the calculated metrics are comparable with those presented in other studies that have evaluated pix2pix networks. In , four apTwo proof-of-concept nuclear molecular imaging in vivo experiments have been conducted in order to demonstrate the efficacy of the method in terms of enhancing anatomical information in functional imaging preclinical studies. For that purpose, two commercial planar radioisotope imaging systems with a pre-installed conventional photographic sensor have been used . The produced X-ray superimposed with the corresponding nuclear image provides an accurate morphological map of the small animal that is used to better identify the organs that the studied compound has accumulated.Future work is oriented in expanding our dataset with data augmentation techniques in order to improve the generalization of our method and hyperparameter tuning in order to further tailor the behavior of the pix2pix network to the final dataset. Taking into account that the primary information comes from photographic sensors and given that small mice used in preclinical studies have similar dimensions and weight (∼20–40 g), geometric and/or photometric transformations may expand the dataset without losing the generalization of the approach, thus providing better performance metrics.


Contact dermatitis is a continuous growing environmental and occupational health problem. It results in high costs for health care systems and the economy due to productivity loss. Moreover, it has a huge impact on the quality of life of patients. The immune response to contact allergy is very complex and not totally elucidated. Recently unique pathways preferentially activated by different allergens were identified. As for a lot of chronic itch disorders, antihistamines are ineffective for allergic contact dermatitis, suggesting a non-histaminergic itch. The precise mechanisms that underlie the development of itch in ACD remain poorly defined. This short review addresses the most recent insights in pruritus in ACD, opening perspectives for future therapies. Contact dermatitis is a common reason of consultation: skin problems provoked by contact with chemicals is a growing environmental and occupational health problem .We distinguish 2 types of contact dermatitis, taking into consideration the pathophysiological mechanism, namely irritant contact dermatitis (ICD) and allergic contact dermatitis (ACD).Irritant contact dermatitis represents 80% of all contact dermatitis cases. It can occur after a single exposure to an irritant or toxic substance, inducing skin damage due to a direct and local cytotoxic effect. Clinical lesions can vary from erythema to a vesicular reaction, or even a caustic burn with necrosis. In case of low grade irritants it will take much longer to observe clinical manifestations. This chronic type of ICD can be observed after accumulative and repetitive exposure to irritative substances such as soap and detergents.The other 20% of cases of contact dermatitis are due to ACD. ACD presents as an itchy eczematous reaction of the skin, occurring hours to days after contact with an allergen. In a chronic phase it presents as erythematous, scaly and lichenified lesions. ACD affects about 20% of the adult general population . A recenAs itch is a dominant symptom in ACD with a huge impact on patient's quality of life, we would like to present a short overview of the current knowledge concerning the mechanisms responsible for itch in patients with ACD. Even if the precise mechanisms that underlie the development of itch in ACD remain poorly defined, every new step in understanding could lead to novel therapeutic approaches.ACD is a delayed type IV hypersensitivity reaction to a hapten or non-protein contact antigen. Its pathophysiology is characterized by 2 phases, which involve both innate and adaptive immune responses . First iIL-23 is a pro inflammatory cytokine from the family of the IL-12, which plays a role in the development of Th17 cells. The Th2/Th22 immune response is the main factor in the development of atopic dermatitis but the Th1 immune response and the Th17/IL-23 signaling pathway could play a role in the transition to a chronic state . IL-23 sIL-25  is produced by epithelial cells and various immune cells. It can induce Th2 cell differentiation and activation suggesting its involvement in Th2-type immune responses.A recent study investigated the contribution of IL-25 in a mouse model of CHS challenged with fluorescein isothiocyanate: the results showed that unexpectedly mast cell- and non-immune cell-derived IL-25 was important for hapten-specific Th17 cell - mediated rather than Th2 cell - mediated inflammation in the elicitation phase by enhancing Th17 related but not Th2-related, cytokines in the skin .These observations demonstrate that ACD is not a single immunological process. There seems to be a mechanistical difference between allergens. This means also that animal models may not be representative of environmental cases of ACD. Moreover, these insights could have therapeutical consequences: targeted-therapy, taking into consideration sensitivity to a specific allergen could enhance efficiency, especially for difficult to treat cases not responding to the classical treatments.Although ICD and ACD share similar clinical characteristics their immunological mechanisms are quite different. In the early stages, ICD and ACD may share cytokines associated with the activation of the innate immune system, but in ICD there is no involvement of antigen-/allergen–specific T cells and the adaptive immune system . Direct Itch is initiated when endogenous or exogenous pruritogens interact with itch receptors or pruriceptors (pruritus + receptor), which resides in the membrane of the nerve endings of primary afferent C fiber somatosensory neurons . There aThe most well defined form of itch is histamine-mediated or histaminergic itch. Mast cells are the key players via release of histamine which activates receptors present on itch-sensory neurons of the dorsal root ganglia (DRG). Mast cell activation results from antigen binding to IgE antibody and cross-linking of the high-affinity IgE receptor, (Fc ε RI). Antihistamines however, are ineffective for ACD like it is the case for a lot of chronic itch disorders such as atopic dermatitis (AD) . ACD is Mast cells are often found in close association with nerve fibers in peripheral tissues . ApparenThe recent study of Meixiong et al. delivers very interesting findings for the scope of this overview: distinct types of mast cell stimuli induce a differential release of mast cell mediators leading to the activation of distinct sets of sensory neurons and itch modalities . Mrgprb2Additionally they observed that the distinct mast cell activation led to activation of distinct neuronal subtypes. Neurons excited by mast cell activation through PAMP9-20 (ligand of Mrgprb2) most consistently overlapped with Mrgpd+ itch neurons, while neurons excited by mast cell activation through anti-IgE overlapped with histamine-sensitive neurons. In itch sensory neurons, H1R expression and Mrgprd expression do not overlap , an indiFinally Meixiong et al. studied the role of Mrgprb2 in ACD: itch behavior was significant reduced in Mrgprb2 -/- mice compared to wildtype (WT) mice for three classical models of ACD . Total CD45+ immune-cell numbers were reduced in Mrgprb2-/- mice compared to WT mice in DNCB-treated mice, these findings are an indication Mrgpr signaling could play a role in immune cell recruitment in ACD. The authors also observed increased levels of PAMP1-20, an MRGPRX2 agonist as well as increased numbers of mast cells in the skin of ACD patients in comparison to healthy controls. All these data suggest a clear role of mrgprb2 (mice) and MRGPRX2 (human) in ACD itch. Also to note is that Mrgprb2-/- animals had a significant reduction in pruritus but still had some residual ACD itch. This could be explained by alternative mechanisms, such as IL-33 activation of neuronal ST2 and T cell release of IL-31 , 25, 27 Analyzing the results of Meixiong et al. important questions may be put forward . Which mCytokines are a broad category of signaling molecules utilized by immune cells and keratinocytes for communication. It has been recognized in recent years that itch sensory neurons also express cytokine receptors and that cytokines may act as pruritogens , 28. Upositu primarily by Th2 and Th1 lymphocytes in a subacute phase continuing during the chronic phase. IL-31 has 2 receptors: oncostatine M-receptor (OCMR) and IL-31 receptor A (IL-31-RA). After a repetitive and long lasting skin process Th2 and Th1 lymphocytes would have produced in situ IL-31 in a sufficient concentration that it could exert his action through 2 pathways according the model of  . ExpressThe same study which showed that the IL-31 levels were significantly higher in patients with ACD showed that the IL-33 serum levels were similar in patients and healthy control patients . Taking Thymic stromal lymphopoietin (TSLP) is an epithelial-derived cytokine and a master initiator of Th2 type inflammation . KeratinLiu et al. could demonstrate in a study comparing mouse ACD models induced by the poison ivy allergen urushiol and the synthetic allergen oxazolone that neutralizing TSLP could significantly diminish scratching behavior in urushiol-challenged mice, suggesting a key role in the itch response of poison ivy ACD . In the Contact dermatitis is a growing environmental and occupational health problem. It results in high costs for health care systems and the economy due to loss of productivity . MoreoveOver the last years we have received new interesting insights into the pathogenesis of ACD: the identification of distinct immune pathways and the activation of these pathways by different allergens . Itch isThe author confirms being the sole contributor of this work and has approved it for publication.The author declares 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.


International air travel plays an important role in the global spread of SARS-CoV-2, and tracing of close contacts is an integral part of the public health response to COVID-19. We aimed to assess the timeliness of contact tracing among airline passengers arriving in Vietnam on flights containing COVID-19 cases and investigated factors associated with timeliness of contact tracing.We included data from 2228 passengers on 22 incoming flights between 2 and 19 March 2020. Contact tracing duration was assessed separately for the time between the date of index case confirmation and date of contact tracing initiation , and the date of contact tracing initiation and completion . We used log-rank tests and multivariable Poisson regression models to identify factors associated with timeliness.p-value = 0.018). Cumulative hazards for successful tracing were higher for Vietnamese nationals compared to non-Vietnamese nationals (p < 0.001).The median duration of interval I and interval II was one (IQR: 1–2) and 3 days (IQR: 2–5), respectively. The contact tracing duration was shorter for passengers from flights where the index case was identified through mandatory testing directly upon arrival  compared to flights with index case detection through self-presentation at health facilities after arrival  (Contact tracing among flight passengers in the early stage of the COVID-19 epidemic in Vietnam was timely though delays occurred on high workload days. Mandatory SARS-CoV-2 testing at arrival may reduce contact tracing duration and should be considered as an integrated screening tool for flight passengers from high-risk areas when entering low-transmission settings with limited contact tracing capacity. We recommend a standardized risk-based contact tracing approach for flight passengers during the ongoing COVID-19 epidemic.The online version contains supplementary material available at 10.1186/s12879-021-06067-x. COVID-19 has spread globally since the beginning of 2020. Within 9 months, SARS-CoV-2 infected over 40 million people and caused over 1 million fatalities worldwide , systemaVietnam has experienced many infectious disease importations during the last 20 years –7, howevWhile contact tracing has proven critical for the containment of COVID-19 transmission in general , there iIn Vietnam, the Government delegated the National Steering Committee for COVID-19 Prevention and Control to implement case finding and contact tracing activities among all passengers of flights where at least one COVID-19 case was detected. This process entailed nation-wide multi-institutional cooperation to contact, test, and quarantine passengers after arrival in Vietnam.During the course of March 2020, Vietnam’s government gradually introduced policies for compulsory SARS-CoV-2 testing and 14-day quarantine for passengers from some designed areas. This included the United Kingdom and 26 Schengen countries on 14 March  and was For this analysis, self-presentation was defined as index cases who presented themselves at local health facilities after arrival due to COVID-19 symptoms or due to recent travel history to affected areas following public awareness campaigns. Mandatory testing was defined as index cases who were tested mandatorily for SARS-CoV-2 at immigration points directly upon arrival.Contact tracing of flight passengers for COVID-19 in Vietnam during 2020 was a complex operation that involved multiple jurisdictions and institutions, including the Ministry of Health (MoH); the National Institute of Hygiene and Epidemiology (NIHE); the Rapid Response Team (RRT) of the National Steering Committee of COVID-19 Prevention and Control; the Civil Aviation Administration and Immigration Bureau; 63 provincial Centres for Disease Controls and Prevention (CDC); and the local police. The MoH assigned the NIHE as the key institution to manage and store data of contact tracing for flight passengers, including SARS-CoV-2 test results. Whenever a laboratory-confirmed polymerase chain reaction (PCR) test was positive, this was immediately reported by the testing facility to the NIHE. The NIHE then passed on the contact information of confirmed cases to the RRT. The RRT contacted and interviewed all COVID-19 cases for their travel history on international or domestic flights within 14 days from date of first symptom onset or date of first positive test result, whichever came first. The RRT then requested the Civil Aviation Administration and Immigration Bureau to provide flight manifests including passenger names, seat numbers, and personal contact details. The RRT shared this information with provincial CDCs and the local police. Local CDC staff and the local police jointly contacted passengers using email, telephone, or social media accounts if available. If necessary, other information such as residential or workplace addresses, information from tourism companies and embassies (for tourists), temporary/permanent residential registration data from local authorities, police department records, and immigration bureau data were also obtained. Furthermore, the MoH issued daily public announcements of flight details on mass media to advise all passengers on these flights to self-present immediately at designated facilities  updated daily by local CDC staff on the national COVID-19 Contact Tracing Management System. All successfully traced passengers were instructed to immediately self-quarantine while awaiting health staff to take a nasopharyngeal swab for SARS-CoV-2 PCR testing. All traced passengers were subsequently transferred to designated quarantine areas regardless of their test result. Passengers who had already left Vietnam were instructed to contact respective health jurisdictions at their current location. Contact tracing of a flight was closed 14 days after the last successful tracing attempt occurred; all passengers not reached at that point were declared lost-to-follow-up. For this analysis, contact tracing completion was marked as the date when the last passenger on the flight was successfully traced.We extracted data for flight numbers, place of departure, arrival dates, total passenger numbers on each flight, passengers’ nationalities, the epidemiological index case detection method of each flight, date of first laboratory confirmation of the index case, date of contact tracing initiation and completion, and final contact tracing status  from the national COVID-19 Contact Tracing Management System.We used frequency statistics to describe the total number of passengers needed to be traced and successfully traced each day. We calculated time intervals between the date of index case confirmation and the date of contact tracing initiation , and the date of contact tracing initiation and date of completion , both overall and separately by method of index case detection (self-presentation vs. mandatory testing). The outcome of interest was the total duration of contact tracing, defined as time between date of index case confirmation and date of completion .We calculated Kaplan Meier estimates to plot the probability of unsuccessful tracing  over time and used log-rank tests to compare the cumulative hazards of successful tracing between both methods of index case detection , passenger’s nationality (Vietnamese vs. non-Vietnamese), and number of passengers per flight requiring tracing . If a passenger remained untraced at date of analysis (31 March 2020), the observation period was considered right censored. In addition, we developed a multivariable Poisson regression model to assess the association between index case detection method and timeliness of contact tracing adjusted for passenger nationality and the number of arriving passengers per plane. Data were analyzed using R version 4.0.2 .n = 1785), while 1.2% (27 passengers) were lost-to-follow-up. The remaining 391 passengers (17.6%) had already transited to other countries at time of investigation . Most passengers were successfully traced . Similarly, the combined time interval between index case confirmation and contact tracing completion for flights with index case detected by self-presentation  was longer than for flights with an index case detected by mandatory testing    and for Vietnamese passengers than for non-Vietnamese nationals .Figure Table We evaluated the timeliness of contact tracing and factors associated with timeliness among 22 flights between 2 March and 19 March 2020, shortly before Vietnam’s government issued the universal test and quarantine policy for all inbound passengers. Our findings suggest that mandatory SARS-CoV-2 testing at arrival can help to reduce the contact tracing duration of passengers upon detection of an index case.This study provides important evidence on integrated contact tracing of flight passengers in a low-middle income country in the midst of the ongoing COVID-19 pandemic.The short tracing duration for passengers on flights with index case detection by mandatory testing at the time of arrival can be explained with the reduced delay in index case detection compared to flights where index cases self-presented, which in turn meant that passengers on these flights had less time to disperse throughout the country and change locations multiple times, which incurred delays. However, this test and quarantine policy by area of departure individually for each passenger  was complex to implement. With SARS-CoV-2 increasingly spreading to more and more countries in March 2020 anyways, it was soon replaced by a blanket mandatory quarantine policy for all incoming passengers irrespective of origin.Although most passengers were successfully traced within 2 days of initiating contact tracing, we still recorded a long tail of unreached passengers over time, indicating a lengthy process to locate some passengers. The reasons for this were manifold and, though reasons for unsuccessful tracing were not recorded systematically, several of them seem to have affected international tourists more than Vietnamese nationals, which might explain the lower cumulative hazards of successful tracing in non-Vietnamese than in Vietnamese nationals. For example, fast-changing locations, lack of local mobile phone contact details, and language barriers were obstacles often experienced by the contact tracers according to our anecdotal experience. Some of these passengers were successfully reached via email, their social media accounts, or tourism companies, while for others the involvement of their embassies or the Vietnamese immigration bureau was required, which added to the delay. Although the MoH issued information of flights with confirmed cases on mass media in both Vietnamese and English, this might not have reached all international tourists, which can explain further delays in this group. Incorrect or missing contact information added complexity and challenges to trace passengers. All inbound passengers from international flights had to complete a health declaration form either via a paper-based or web-based version, which was not always done correctly and completely. While the web-based data was directly linked to the passengers’ management system, paper-based forms had to be entered manually by the Civil Aviation Administration and Immigration Bureau. Due to the large daily volume of paper-based records at that time, data entry errors might have occurred more frequently during high workload days.Our results suggest that contact tracing was more time-consuming with increasing passenger volume of that flights, which makes sense in light of the sudden overload of the system through large volume flights. Furthermore, all flights with medium and high volumes were international connections carrying large numbers of non-Vietnamese nationals. Collecting passenger details from foreign airlines also took longer because of differences in time zones and the need to convince the airline companies to hand over passenger lists, which often required to issue official request letters.Challenged by a surge of passengers needed to be traced, the contact tracing process in Vietnam adapt, as shown by the increasing number of passengers successfully traced. However, this adaptation did not seem to have been enough during days with a high workload, since more than half of the passengers needed to be traced were not reached during these days. While contact tracing was a crucial component for preventing the spread of COVID-19 in the community , it is iWe acknowledge several limitations of this study. Firstly, available data were recorded in days instead of hours, which adversely impacted the accuracy of our estimates. Secondly, for flights with index cases detected by mandatory testing, some passengers were already tested and quarantined upon arrival at the same time as the index cases. However, we could not exclude such passengers from our analysis due to data unavailability but we estimate the number to be relatively low since the list of designed high-risk areas, which required direct quarantine upon arrival only of those passengers who started their journey from a high-risk area but not of all passengers on that plane, was only gradually expanded (see Additional file Contact tracing among flight passengers in the early stage of the COVID-19 epidemic in Vietnam was timely, although delays occurred on high workload days. Mandatory SARS-CoV-2 testing at arrival may reduce contact tracing duration and should be considered as an integrated screening tool for flight passengers from high-risk areas when entering low-transmission countries with limited contact tracing capacity. We recommend a standardized risk-based contact tracing approach for flight passengers during the ongoing COVID-19 epidemic.Additional file 1. Public health interventions to prevent transmission from flights in Vietnam.Additional file 2. Timetable from arrival to contact tracing completion for 22 tracing flights, March 2020, Vietnam.