The present study aims to delineate the role of preexisting depression for changes in common mental health problems during the COVID-19 pandemic.n = 6,694) and prospectively followed up at the peak of the second COVID-19 wave .Using mixed-effects linear regression models, we analyzed data on the course of depressive  and anxiety  symptoms as well as loneliness  in a subset of the Socio-Economic Panel Study, a large and nationally representative household panel study from Germany. Participants were assessed during the first COVID-19 wave in Germany (March 31 to July 4, 2020; Overall, anxiety and depressive symptoms decreased, whereas loneliness increased from the first to the second COVID-19 wave. However, depressive symptoms increased and the surge in loneliness was steeper in those with versus without clinically relevant depressive symptoms in 2019 or a history of a depressive disorder before the COVID-19 pandemic. Anxiety symptoms remained stable throughout the pandemic in individuals with versus without clinically relevant depressive symptoms in 2019. Pre-pandemic depression was associated with overall higher depressive and anxiety symptoms and loneliness across both assessments. The stringency of lockdown measures did not affect the results.Our findings suggest that individuals with a history of depressive symptoms before the COVID-19 pandemic are at increased risk to experience an escalation of mental health problems due to the COVID-19 pandemic. Therefore, they might particularly profit from targeted prevention and early intervention programs. Depressive symptomatology is prevalent among the general population and causes major psychological, social, and economic burdens , 2. IndiThis vulnerability to stressful events in depression may become particularly relevant in times in which individuals are faced with an inevitable stressor such as the COVID-19 pandemic that may threaten mental health. This assumption is corroborated by reports of increases in experienced distress, anxiety, depression, and loneliness from the time before the pandemic to the first wave of the pandemic in Germany , 13 and It is particularly plausible to assume that especially individuals affected by elevated depressive symptoms before the pandemic would show unfavorable changes in mental health during the COVID-19 pandemic. Recent studies on the effect of depression on mental health during the COVID-19 pandemic, however, revealed inconclusive results. Current data collected during the initial phase of the COVID-19 pandemic indicated that previous or current depression was associated with higher depression, anxiety, loneliness, and COVID-19-related distress during the beginning of the COVID-19 pandemic , 26. In a priori hypothesis of whether preexisting depressive symptomatology is associated with unfavorable changes in mental health during the COVID-19 pandemic. Therefore, we analyzed the role of depressive symptoms in 2019 and a history of a diagnosed depressive disorder (as derived from the regular SOEP waves before the pandemic) for changes in depressive and anxiety symptoms as well as loneliness during the COVID-19 pandemic . Elucidating the role of depression in mental health during an enduring stressful situation, such as the COVID-19 pandemic, may help to advance our understanding of the development and chronicity of depression and related mental health problems as well as inform public health and clinical approaches to prevention and intervention.To close this research gap, we used data from the Socio-Economic Panel Study (SOEP), a nationally representative panel study from Germany. A subsample of SOEP participants (SOEP-CoV) was asked about depressive and anxiety symptoms as well as loneliness during the first COVID-19 wave (March to July 2020) and 6 months later, during the second COVID-19 wave (January to February 2021). In this publication, we aimed to test our n = 6,038), that is, from January 18 to February 15, 2021 [We used data from a subset of SOEP, a nationally representative household panel with approximately 30,000 participants in 15,000 German households assessed since 1984 (v36) . In 202015, 2021 . As can Sociodemographic information, such as age, gender, and education, was assessed during the COVID-19 assessment or derived from previous assessment waves. Education was stratified as primary, secondary, and tertiary based on the highest level of education or vocational training according to the Comparative Analysis of Social Mobility in Industrial Nations. The equivalized household income included all disposable monthly household incomes, including all types of after-tax and transfer income. Differences in household size are considered by equivalization, that is, divided by the square root of the number of household members.Loneliness. In 2020 and 2021, loneliness during the past 2 weeks was assessed with the German version of the three-item UCLA Loneliness Scale [ss Scale  and ratess Scale , our stuDepressive and anxiety symptoms. In 2020 and 2021, depressive symptoms over the past 2 weeks were assessed with the two-item Patient Health Questionnaire-2  and anxiety was assessed with the two-item Generalized Anxiety Disorder-2  questionnaire [ionnaire –33. Simiionnaire , 32, in Depression diagnosis. In 2019, individuals were asked to indicate whether a doctor had ever diagnosed a depressive disorder.Depressive symptoms in 2019. In 2019, depressive symptoms were assessed using the PHQ-2. PHQ-2 scores of 3 and above indicate a depressive disorder with a sensitivity of 79% and a specificity of 86% [y of 86% .Restrictions due to public health measures in 2020. In 2020, 14 forms of restriction measures that have been suggested to disrupt self-regulated and psychologically relevant behavior of individuals were systematically documented for each of the 16 German federal states on a day-by-day basis  by the Leibniz Institute for Psychology Information . Each tAll analyses were conducted using mixed-effects linear regression models with repeated measurement occasions (Level 1) nested within persons (Level 2), nested within federal states (Level 3). First, we analyzed changes in depressive and anxiety symptoms as well as loneliness from 2020 to 2021. Specifically, we regressed the score of the respective outcome  on a timing variable (Level 1), coded with 0 at the first wave and coded with 1 at the second wave of the SOEP COVID-19 assessments. To investigate the role of (a) pre-pandemic depressive symptoms (PHQ-2 score in 2019) and (b) a history of a depressive disorder (no vs. yes) for changes in the respective outcome from 2020 to 2021, an interaction term between the respective predictor  and the timing variable was included in the respective model. Significant interactions were probed using simple slope analyses  to evaluβ = −0.02, p = 0.020; anxiety symptoms: β = −0.03, p < 0.001; see Supplementary Table S1). Conversely, loneliness increased during the same period .As can be seen in p < 0.001; see Supplementary Table S2). Moreover, changes in depressive symptoms during the pandemic varied as a function of pre-pandemic depressive symptoms in 2019 . To assess the direction of interaction in greater detail, we distinguished between individuals above and below the clinical cutoff PHQ-2 score for a potential depression diagnosis in 2019. Post hoc simple slope analyses revealed that individuals above the cutoff score experienced an increase of depressive symptoms from 2020 to 2021 (p < 0.001), whereas individuals below the cutoff score experienced a decrease (see the upper-left panel of p < 0.001).Pre-pandemic depressive symptoms were associated with overall higher levels of depressive and anxiety symptoms as well as loneliness in 2020 and 2021  predicted a steeper increase in loneliness from 2020 to 2021 (pre-pandemic depressive symptoms × time: p > 0.05). However, as depicted in the upper panel of p > 0.05), but decreased in individuals below the cutoff score (p < 0.001).Changes in anxiety symptoms did not vary significantly by pre-pandemic depressive symptoms . Most interestingly, depressive symptoms increased from 2020 to 2021 in individuals with (p = 0.03), but decreased in individuals without a pre-pandemic depression diagnosis . Change in anxiety during the pandemic did not differ significantly between individuals with versus without a pre-pandemic depression diagnosis (p > 0.05).A pre-pandemic depression diagnosis was associated with overall higher levels of depressive and anxiety symptoms as well as loneliness in 2020 and 2021 .The stringency of lockdown restrictions in 2020 did not interact significantly with pre-pandemic levels of depressive symptoms or a pre-pandemic depression diagnosis in predicting changes in depression, anxiety, or loneliness from 2020 to 2021 (all In the present study, we examined the effect of pre-pandemic depression on the course of depressive and anxiety symptoms as well as loneliness during the COVID-19 pandemic in a large probability sample of adults from Germany. We found that depressive and anxiety symptoms decreased, whereas loneliness increased from the first to the second wave of the COVID-19 pandemic in Germany. However, these symptom changes varied as a function of pre-pandemic depressive symptoms in 2019 and a diagnosed depressive disorder before the COVID-19 pandemic: depressive symptoms increased and the surge in loneliness was steeper in those with versus without clinically relevant depressive symptoms in 2019 or a history of a depressive disorder before the COVID-19 pandemic. Changes in anxiety symptoms did not vary as a function of a pre-pandemic depressive disorder. However, throughout the pandemic, anxiety symptoms remained stable in individuals with but declined in individuals without clinically relevant depressive symptoms prior to the pandemic. The stringency of lockdown measures did not affect the results.early stage of the COVID-19 pandemic improved in individuals with and without pre-pandemic mental health problems [Taken together, our findings suggest that the long-term course of depressive and anxiety symptoms as well as loneliness during the COVID-19 pandemic is less favorable in vulnerable individuals with versus without pre-pandemic depression. These results are in line with evidence that depression relates to less favorable responses to inevitable and uncontrollable/unpredictable stressors , 37. Howproblems . These fOur finding that pre-pandemic depression predicted a greater increase of depression and loneliness during the pandemic is consistent with previous evidence that depression prospectively predicts higher loneliness and depressive symptoms , 38. TheParticular strengths of this study are the prospective-longitudinal design, the inclusion of pre-pandemic data, and the use of a probability sample of a nationally representative household panel, which have been highly recommended as it reduces the risk of sampling and recall biases , 42. In In the present study, individuals with elevated depressive symptoms or a history of a diagnosed depressive disorder were identified as a risk group for an unfavorable course of mental health problems during the COVID-19 pandemic. The observed persistence of anxiety symptoms as well as the increases in depressive symptoms and loneliness in this group might increase the risk for the clinical manifestation, exacerbation, or chronicity of depressive symptomatology and related somatic and mental health sequels. In particular, loneliness—a common concomitant feature of depression—has been associated with an increased risk for several mental disorders and somatic diseases in general , 9, 43.


Among terrestrial vertebrates, only crown birds (Neornithes) rival mammals in terms of relative brain size and behavioural complexity. Relatedly, the anatomy of the avian central nervous system and associated sensory structures, such as the vestibular system of the inner ear, are highly modified with respect to those of other extant reptile lineages. However, a dearth of three-dimensional Mesozoic fossils has limited our knowledge of the origins of the distinctive endocranial structures of crown birds. Traits such as an expanded, flexed brain, a ventral connection between the brain and spinal column, and a modified vestibular system have been regarded as exclusive to Neornithes. Here, we demonstrate all of these ‘advanced’ traits in an undistorted braincase from an Upper Cretaceous enantiornithine bonebed in southeastern Brazil. Our discovery suggests that these crown bird-like endocranial traits may have originated prior to the split between Enantiornithes and the more crownward portion of avian phylogeny over 140 Ma, while coexisting with a remarkably plesiomorphic cranial base and posterior palate region. Altogether, our results support the interpretation that the distinctive endocranial morphologies of crown birds and their Mesozoic relatives are affected by complex trade-offs between spatial constraints during development. Two mheropods ,21, or vthyornis ,10), shathyornis .Archaeopteryx and the most crownward portion of the avian stem lineage [Here, we describe a partial braincase from an Upper Cretaceous (approx. 80 Ma) avian bonebed in southeastern Brazil . The braincase is nearly undistorted  consists of a diminutive basicranium .Neuquenornis [Yuornis [Zhouornis in which these structures are more similar in size  among the osteological components, similar to other adult or subadult enantiornithine specimens , suggestuenornis  but unli[Yuornis  or Zhououenornis ) is visiuenornis .Figure Archaeopteryx, Cerebavis and many crown birds [The preserved endocast—visualized with micro-computed tomography (μ-CT) imaging (see Methods)—comprises impressions from the external surfaces of most of the medulla, parts of the cerebellum, and the ventral portion of the optic tectum . We intewn birds —the degrArchaeopteryx [Cerebavis [Cuculus canorus and Haematopus ostralegus)   , contras. 3The new braincase MPM-334-1) combines plesiomorphic dinosaurian traits  with large semicircular canals and a flexed brain that are remarkably similar to those of extant birds  or other parts of the brain (e.g. the cerebrum in Corvus) appear to push the endosseous labyrinth ventrally during development , indicating either independent strong ventralization within enantiornithines or potential reversals to less ventralized morphologies along the line towards modern birds  and ornithes , which cIchthyornis suggests that stem ornithuromorphs retained a plesiomorphically unflexed brain morphology until very late in their evolutionary history [Ichthyornis could underlie the unflexed nature of its brain as seen in many Aequornithes (see the above discussion).Our observations suggest two alternative macroevolutionary scenarios in the history of avian endocranial evolution. On the one hand, the suite of endocranial transformations common to MPM-334-1 and many extant birds  may have arisen among relatively small-bodied terrestrial and/or arboreal taxa prior to the origin of Ornithothoraces, while some crownward stem birds and neornithines (such as the aforementioned waterbirds) reverted to a condition resembling the plesiomorphic pre-ornithothoracine endocranium. On the other hand, an alternative scenario suggests that the same suite of endocranial features could represent homoplasies that arose multiple times throughout the evolutionary history of birds: within Enantiornithes such as in MPM-334-1, and again somewhere along the most crownward portion of the avian stem lineage (e.g. crownward of Hesperornithiformes). The latter scenario is consistent with the considerable degree of homoplasy already reported between enantiornithines and ornithuromorphs across the skeleton, including the independent acquisition of a rectricial feather fan , a tooth history . That saRegardless of the precise phylogenetic origin of these traits among Mesozoic birds, our results and earlier work collectively suggest that the flexion of the avian brain is closely related to the orientation of the occiput , causingMPM-334-1 illustrates that a substantial degree of brain flexion and ventralization of the foramen magnum had already evolved among enantiornithine birds, indicating that important transformations towards a modern bird-like central nervous system and sensory organs arose early in avian evolutionary history. Conversely, other regions of the skull of MPM-334-1, especially the cranial base, retain a plesiomorphic dinosaurian configuration, illustrating that endocranial evolution in enantiornithines was somewhat disconnected from the morphological evolution of the remainder of the skull. Overall, the discovery of MPM-334-1 supports a more complex scenario for the evolution of the avian skull and central nervous system than had previously been understood, with key features of the modern avian endocranium evolving much earlier than what was formerly thought. Future discoveries of three-dimensionally preserved braincases from other Mesozoic birds, and their incorporation into quantitative analyses such as those presented here, will prove crucial for furthering our understanding of the origin and early evolution of the avian skull, brain and sensory systems.. 4 (a)vizo Lite (9.2). Digital mesh cleaning was conducted using Geomagic (2013). Final imaging of the volumes was conducted using Blender and Avizo Lite (9.2).MPM-334-1 was scanned using a GE Phoenix Nanotom M at the Molecular Imaging Center of the University of Southern California (USC). MPM-334-1 was scanned at 9.99 µm voxel size, 125 kV, 200 mA, exposure time 750.36 ms, averaging two frames and skipping one frame, 360 degrees rotation 1440 frames and 0.1 mm Cu + Cu filter. The scans were initially reconstructed using GE phoenix datos|×2 2.3.3.160. The three-dimensional reconstruction of the skull was generated in A (b)n = 13), together with a large sample of crown and stem birds (n = 314) sourced from several online repositories . We placed four landmarks on the three-dimensional meshes of the skull for each species: two within the parasphenoid rostrum and two at the dorsal-most and ventral-most points of the internal rim of the foramen magnum, using Stratovan Checkpoint (v.2019.03.04.1102) and its built-in functions to calculate the angle between the two vectors described by the four landmark coordinates.To gain insight into the degree of ventralization of the foramen magnum in MPM-334-1, we measured the internal angle between the parasphenoid rostrum and the foramen magnum in the new specimen and compared it with a broad phylogenetic sample of skulls from non-avian dinosaurs, with a focus on near-avian theropods (


Implementing evidence-based recommendations with the option of patient-individualised and situation-specific adaptations in telerehabilitation may increase adherence with improved clinical outcome.As part of a registry-embedded hybrid design (part 1), digital medical device (DMD)-usage in a home-based setting was analysed in a multinational registry. The DMD combines an inertial motion-sensor system with instructions for exercises and functional tests on smartphones. A prospective, single-blinded, patient-controlled, multicentre intervention study (DRKS00023857) compared implementation capacity of the DMD to standard physiotherapy (part 2). Usage patterns by health care providers (HCP) were assessed (part 3).2 = 44.9, p<0.001). Intention-to-treat-analysis (part 2) revealed DMD-users to have significantly higher adherence to the rehabilitation intervention compared to the matched patient-control-group . DMD-users performed recommended exercises at home with higher intensity (p<0.05). HCP used DMD for clinical decision making. No adverse events related to the DMD were reported. Adherence to standard therapy recommendations can be increased using novel high quality DMD with high potential to improve clinical rehabilitation outcome, enabling evidence-based telerehabilitation.Registry raw data  were analysed from 604 DMD-users, demonstrating clinically expected rehabilitation progression post knee injuries. DMD-users performed tests for range-of-motion, coordination and strength/speed enabling insight to stage-specific rehabilitation (χ Telerehabilitation refers to rehabilitation programmes delivered by electronic services such as medical apps. This technology can facilitate health care access and improve individual adherence to recommendations in a home-based setting, especially in the early phases of rehabilitation. We investigated key aspects of a sensor-guided medical app used for knee rehabilitation after surgery in three separate studies. We compared usage patterns, clinical effectiveness, and safety from 604 nationwide users and found that the sensor and medical app are able to measure correct values in the rehabilitation progress when used only by patients at home. Using the tests suggested by the app was safe. Patients with the app in the home environment were more likely to stick to recommendations of exercises given by doctors and physiotherapists and train harder compared to those only undertaking physiotherapy. Doctors and physiotherapist used sensor data and the medical app to decide what type of exercise a patient should perform. Our data show that telerehabilitation, and medical apps in particular, may become a game-changer for individualised and functional approaches to orthopaedic rehabilitation programmes. Telerehabilitation has a high potential to improve adherence and clinical outcomes in guideline-based rehabilitation programmes. Implementing evidence-based rehabilitation recommendations with individualised and situation-specific adaptations may increase adherence to health care providers’ recommendations at home with subsequent improved clinical outcomes . In orthFurther, telemedical applications may ensure correct performance of exercises. Validated sensors  that areIn the initial phase of rehabilitation after acute knee joint injuries and/or after surgical interventions, active and passive knee joint mobility becomes relevant, while later functional stability, coordination, and strength become necessary before dynamic and speed exercises will be possible –10.Adherence is the degree to which a person’s behaviour meets the agreed-upon recommendations of a health care provider . There iWe here present three different perspectives of the first DMD for knee rehabilitation. First, we sought to determine the usage patterns, measurement of clinical effectiveness and safety of the DMD in real-life from registry data. Next, we tested the prespecified hypothesis of its effect on clinical efficacy (adherence) and safety. Finally, we present the usage patterns and requirements of health care providers for a DMD as add-on to standard rehabilitation. All data stem from a registry-embedded hybrid design.ORthelligent telematic rehabilitation System OutcoME” study, ORSOME-study, DRKS00023857) on patients after surgical intervention for knee injuries. The prespecified primary endpoint of superiority of adherence to rehabilitation was quantified from a score deducted from a beforehand validated adherence questionnaire (ADREHA-score) and compared to a similar patient control group who received standard physiotherapy. In the third part, involved health care providers were requested to provide feedback on usage, adherence, and clinical outcome in patients using the telerehabilitation.A registry-embedded hybrid design of DMD in rehabilitation in German-speaking countries in real life was performed. Approval was obtained by the local ethics committee prior to trial commencement . In the first part, a systematic data analysis of primary raw data of all users of a DMD-registry was conducted. This was followed by a prospective, single-blinded, patient-controlled, and formally multicentre study  is a validated Class I medical device . It consFrom January 2018 to September 2020, 33,057 records of 604 patients who used a DMD-system for any indication were stored in a comprehensive multinational registry . After plausibility-driven  data cleanings, the remaining 10,311 values were further analysed. To exclude intra-week fluctuations and repetitively performed tests in order to record best individual measurements at acquisition dates, only the best value per week was considered for further analysis.After performance of each test, demographic data, medical history, usage data, measurement results of each test performed, and information about sensation of pain on the injured leg were recorded. Pain was quantified by a visual analogue scale ranging from 0–10  . InformaBetween December 2020 and February 2021, consecutive patients (n = 308) from the registry, who had received a knee orthosis, received a one-time invitation to participate in an online survey to give information on their individual 6 months rehabilitation after knee surgery. This procedure was in accordance with the corporate policy, EU-GPDR and individual consent to data usage and protection prior to using the DMD. Diagnosis data coded according to the International Classification of Disease (ICD) were available for all patients, and diseases were classified accordingly. 30 responded to the survey and gave their digital informed consent. The study was approved by the local ethics committee (Eth-53/20).Inclusion criteria for analysis were: DMD-usage (≥ 1 test) or usage of knee orthosis treated within 6 months post-surgery, and informed consent. Exclusion criteria for analysis were: total and /or plausible answers <80%, or DMD-usage in the patient control group. The dataset retrieved from the prospective ORSOME-study contains all patients who made an assignment to one of the two treatment groups after surgical treatment of a knee joint injury or for knee joint damage or instability in an intention-to-treat-analysis (ITT).The following questionnaires were used:- Medical history and preinjury level of sports activity: International Knee Documentation Committee (IKDC) forms  - activity and return to activities of daily life: PAQ 50+  - restrictions related to knee injury: ACL-RSI Knee Score   and subj- QoL: KOOS QoL items - adherence: ADREHA , give their actual condition and the time interval since intervention.Health care providers (HCP) listed in the company’s data bank as prescribers or professional users of any of the company’s products received a one-time invitation to participate in an anonymised digital survey regarding the DMD. Fields of interest were: individual background, usage patterns, current and future indications, subjective assessment of the DMD, their judgement on area of application, perception of patients’ adherence and clinical outcomes and to document the benefits. All ratings by HCP were given on a scale from 0–10.As there are no measurement instruments available for assessing adherence to rehabilitation for the target population of a cohort of patients undergoing rehabilitation after knee joint injuries or for chronic knee injuries in German-speaking countries and the corresponding cultural area, a standardised adherence score was derived from existing and validated components in German language. The questionnaire was tested and internally validated following the COSMIN guideline  accordinData was collected and analysed in a GCP-compliant manner, in accordance with the ethical principles of the Declaration of Helsinki and in compliance with all EU regulations and German legislation.Statistical analyses of the register data bank (part 1) were performed blinded from the database used for exploratory analysis. A range data plausibility check was performed for all independent and dependent outcomes; data was cleaned accordingly. Measurement results of the affected leg were analysed in the pre- and postoperative rehabilitation course for each test only for patients who provided their—planned or actual—date of intervention. Data of patients, who performed the corresponding test postoperatively with at least one repetition during the first 12 weeks, were evaluated for individual temporal courses. Missing or unclear values were not replaced. A minimum of 80% data completeness for key variables was prerequisite for subsequent analysis.Data from the ORSOME study (part 2) were analysed in an ITT-analysis with the prespecified endpoint of increased adherence in the treatment group compared to the patient control group measured by the previously validated ADREHA-score. For details, please see 2) was used to estimate the relative chance of usage and 95% CIs are given. To analyse the frequency of use with respect to time to usage, “time to 50% of patients” (t50) and “time to 75% of patients” (t75) were analysed. Statistical significance is assumed if the null hypothesis can be rejected with a significance level of p ≤ 0.05. IBM SPSS Statistics  was used for analysis.For part 1–3, all continuous variables were examined for normal distribution using Q-Q plots. All metric data is non-normally distributed, presented as median with corresponding quartiles , and compared with Mann-Whitney-U tests. Bivariate non-parametric correlation (Spearman) was used to examine the internal validity of the individual questions used for the ADREHA-score in relation to each other and in relation to the ADREHA-score. Cumulative start to usage of the test categories during follow-up time was univariately evaluated by Kaplan-Meier analysis (log-rank test) and time to test for the earliest test within a category was chosen for analysis. Likelihood ratio chi-square , whereas 20% (n = 122) of the DMD-users were treated only conservatively. Patients started using the DMD at three different time periods related to surgical interventions in home-based settings: (I) 1 year to 8 weeks pre-operatively, (II) peri-operatively (immediately pre- and up to 8 weeks post-surgery), and (III) >3 months post-operatively , range of motion was used by nearly all patients see .2 46 for group comparison, p<0.001, log-rank, Patients started with tests for ROM in the earliest rehabilitation phase, followed by coordination after intervention, whereas strength / speed were started significantly later  or 75% of patients (t75) had used single tests in the early rehabilitation phase (16 weeks), a much more differentiated pattern can be observed with regards to patient heterogeneity. For tests of ROM and coordination, t50 was 1 week and t75 was 2 weeks, similarly for coordination (t50: 2 weeks and t75: 4 weeks), indicating a rather homogenous rehabilitation progress for all patients for these tests. Interestingly, for the dynamic challenges (strength and speed) a different pattern (t50: 3 weeks and t75: 6 weeks) was observed, indicating that a small patient group had performed these tests safely at unexpectedly early timepoints. More than a quarter had not even performed dynamic tests up to 16 weeks.Range of motion objectively quantified with the DMD increased during the course of postinterventional rehabilitation , mainly for the dynamic tests  at the beginning and after 6 weeks . No adverse events were related to the DMD.Of 31 patients consented to participate in the survey regarding adherence to health care providers’ recommendations, 30 patients answered the questionnaires. After the exclusion of 3 patients , 27 data sets were available for analysis  of the DMD-users confirmed the benefit of monitoring their rehabilitation outcome as positive feedback to increase motivation. No adverse events related to DMD-usage in a cumulative 104 months DMD-usage period confirmed the safety of home-based use.Making an appointment with their physical therapist was rated as rather hard  for all joints of the lower extremity  with appr. 20 (5–50) DMD-prescriptions / HCP during the last year. DMDs had been prescribed or recommended from adolescents (13–17 years) up to elderly (> 65 years) patients. The settings were postoperatively (48% of HCP), perioperatively (48% of HCP), and merely conservatively (4%) as home-based use, 43% had been for acute and 19% for chronic orthopaedic conditions, as well as for neurological (n = 3) or muscular disease (n = 6). 45% of HCP already used the ORS and 57% saw the potential for research to generate objective quantifiable data  or as instrument to influence clinical decision making , whether patients can return to work or sports (90% of HCP), optimising individual therapy schedules (81% of HCP), identifying further therapy needs (67% of HCP) and taking influence on patients’ rehabilitation , “Orthelligent” offers the additional possibility of intraindividual comparison  and interindividual comparison to healthy individuals of an amateur or competitive level and to patients in different stages of rehabilitation.Choice of different exercises and testing options of the DMD vary depending on the period before or after surgical intervention, or mere conservative treatment. The pattern presented here clearly shows that the individual tests of the Orthelligent are adequately used by patients at home, depending on their respective individual situation. Furthermore, data of this study support the relevance for an individualised and functional rather than time-dependent rehabilitation programme as currently implemented due to lack of large-scale real-world data. In conclusion, digital medical devices offer the opportunity to serve as a fact-based game-changer for optimal guidelines in rehabilitation programmes.Digital medical device-usage was predominantly pain-free and safe in-home use. This is in line with similar results about the safety of other digitally supported rehabilitation programmes in orthopaedics ,37. One Since the exercises guided by and the test values assessed by the DMD are not specific to rehabilitation after ACL reconstruction, it is not surprising to find health care providers applying the DMD for patients with acute and chronic conditions. The DMD was used predominantly for clinical indications for all joints of the lower extremity, for postoperative, conservative, or preoperative indications, and acute or chronic joint disease, as well as for neurologic or muscular diseases. Health care providers used the ORS in children, adolescents, adults, and also elderly patients (> 65 years). Furthermore, specialised service providers see the benefit of DMD and already use it for a larger group of indications, which goes far beyond the exemplary patient collective shown here .The DMD with measurement functions used in this study offers the potential for a) objective quantitative monitoring of lower extremity function and rehabilitation progress, relevant for health care providers in clinic and research, b) decision making in adjusting rehabilitation measures or giving fact-based recommendations, or c) quality assurance of certain rehabilitation programmes. Planning and controlling the implementation of evidence-based rehabilitation steps are preconditions for individual function- and ability-based control of rehabilitation progress instead of rigid time-based rehabilitation protocols and would constitute a major step forward. This may lead to an adaptation of rehabilitation phases by supporting well-trained and proficient or hesitant and anxious patients. At the same time the risk of a too fast and therefore risky rehabilitation can be avoided, and the rehabilitation programme can be adapted to the individual needs / requirements.We here present a new validated tool (ADREHA-score) for quantification of adherence to rehabilitation recommendations for the middle European culture in German, automatically calculating individuals’ adherence score. Further research is needed to test its validity for other indications and settings or its individual scoring in clinic or research.The data set provided from the server of the DMD shares the strength and limitations of raw data sets primarily intended for non-scientific purposes. The data of more than 30.000 data sets from 604 patients used for originally intended use (post ACL-reconstruction rehabilitation in athletes) as well as other indications  and extended user circles (adolescents and the elderly) show the potential of DMD-usage as an add-on to standard physiotherapy in real life examined in a study population representative of the target population . This fiHowever, no assumptions can be made regarding individuals, who for any reason did not consider usage of this DMD suitable or helpful, or discontinued usage for any reason, be it discomfort or fast rehabilitation. Furthermore, response rate was relatively low in one study part. It is not clear from the evaluation whether and to what extent the patients may have already had pain prior to testing or, if applicable, whether testing was not performed in the case of pre-existing pain. Although designed as a prospective, blinded and controlled study with a prespecified primary endpoint and meticulous attention to confounding variables, an improved clinical outcome as a result to increased adherence can be deducted from clinical experience and is supported by the body of evidence. Still, the underlying causal relation has not been proven in this study and will need to be determined in a large-scale RCT considering sufficient numbers of different indications to confirm our first findings on increased adherence in a relatively small number of patients. Furthermore, prospective trials will be needed to clarify to which extent telerehabilitation might replace, essentially modify, or reduce the frequency of classic standard rehabilitation.Well-designed digital medical devices increase adherence to health care providers’ recommendations regarding rehabilitation programmes and are safe to use. Additionally, valid and reliable mobile measurement tools for quantification of complex functional movements offer substantial value for different health care provider groups. Implementing novel digital technologies may well be a key solution for functional rehabilitation protocols and programmes in the future.S1 FileFig A: Flowchart registry data analysis (part 1) and ORSOME study (part 2); n, number of patients; ITT, intent-to-treat. Fig B: Begin of use at three different time periods related to surgical interventions: (I) 1 year to 8 weeks pre-operatively, (II) peri-operatively (immediately pre- and up to 8 weeks post-surgery), and (III) >3 months post-operatively. Fig C: Kaplan-Meier curve for begin of usage of the DMD for rehabilitation within 16 weeks after knee surgery by test categories (A-C). CI, confidence-interval; n, number of patients. Fig D: Postoperative progression of FIT-Index after surgical intervention in 113 patients. Table B: ADREHA-Score, patient assessment of adherence score elements. Main components of ADREHA-score in arbitrary units (in %) on a scale of 0–10; median [IQR]. Fig E: Benefit of DMD for A) patients and B) health care providers from health care providers perception(DOCX)Click here for additional data file.S1 ADREHA Questionnaire(DOCX)Click here for additional data file.


Anti-N-methyl-D-aspartate receptor (NMDAr) antibody encephalitis is an autoimmune disorder characterized by reduced synaptic activity of the NMDAr due to circulating antibodies that target the NR1 subunit. Few cases of anti-NMDAR encephalitis during pregnancy have been described. The permeation of anti-NR1 antibodies through the placenta can be instrumental in the development of complications in newborns. We describe a case of a young woman suffering from anti-NMDAR encephalitis during the first trimester of pregnancy and focus on diagnostic and therapeutic management. We aim to highlight the positive maternal and fetal outcome and focus on diagnostic and therapeutic management.2A 29-year-old woman in the 7th gestational week came to our observation for the sudden onset of continuous, ongoing, focal motor seizures involving the right side of the face. According to her past medical history, in the previous seven days, the patient had presented several episodes of emotional liability with sudden changes in her mood and behavior . The medical history was negative for any significant comorbidity.At admission to the Emergency Room, during the neurological evaluation, the patient showed continuous (lasting > 60 min), stereotyped, rhythmic muscle jerks involving the right labial commissure and sialorrhea. The patient was fully aware and did not show other focal neurological signs. The patient underwent a video-electroencephalogram (video-EEG) recording, which showed continuous high-amplitude rhythmic 3–5 Hz slow waves and sporadic biphasic sharp waves over the left fronto-centro-temporal derivations . A diagn3) with increased protein (70.5 mg/dl) and normal glucose levels. A polymerase chain reaction assessment of neurotropic viruses and the search of oligoclonal bands were negative. However, the antibodies panel for AE revealed high levels of anti-NMDAR antibodies in the cerebrospinal fluid, a finding confirmed in the serum , with normal global cognition.During the following weeks of pregnancy, the patient did not experience any relapse of the neurological and psychiatric manifestations. At the 39th week of gestational age, natural delivery was performed. At the delivery, the newborn presented normal birth weight (2.800 kg) with APGAR 9–10 at the first and the fifth minute. The newborn did not present any sign of fetal distress with no major or minor malformations. The patient decided to avoid breastfeeding. At the follow-up visit, performed 3 months later, the patients did not show abnormalities at the neurological examination. An MRI scan with contrast of the brain indicated complete resolution of the neuroradiological anomalies. The EEG was also unremarkable. At the new neuropsychological evaluations, only attention deficits with normal global cognition were observed. The patients started a slow down-titration of the ASM therapy with the aim to stop the treatment in the following 3 months. In addition, a follow-up total-body CT scan with contrast, pelvic ultrasonography and breast RX were performed, all negative.3Anti-NMDAR encephalitis is one of the most frequent autoimmune epilepsies (AE) during pregnancy The diagnostic and therapeutic management of anti-NMDAr encephalitis during pregnancy is particularly challenging due to the possible fetal side effects of diagnostic and therapeutic interventions. The diagnosis of AE is clinical Around 55% of women suffering from AE have an ovarian teratoma, but other tumors and hematological diseases have also been described. As reported in Concerning therapy, it is well known that some ASMs and immunomodulatory treatments can have teratogenic and toxic effects. Evidence indicates that certain ASMs, especially first-generation drugs  are associated with increased rates of congenital malformations  as well as with newborn distress Even though seizures in AE are characteristically resistant to ASMs, these drugs maintain a role for symptomatic management. A recent study Immunomodulatory treatment with systemic corticosteroids seems to be safe during pregnancy. Indeed, pregnant women receiving corticosteroids usually show a low risk of developing major congenital malformations, even though premature rupture of amniotic membranes and low birth weight babies may occur In our case, the treatment with steroids and PLEX produced complete relief of the symptoms, good clinical response, and no evidence of newborn’s malformation. In line with previous reports , treatme4Our case describes the diagnostic and therapeutic management of a patient suffering from anti-NMDAr encephalitis during pregnancy. Immunomodulatory treatments along with ASM have played a key role in control of focal-to-bilateral seizures. The prompt diagnosis and the adequate treatment led to a complete recovery of the mother and a good newborn outcome.5Written informed consent was obtained from the patient for publication of this case report and any accompanying images. The paper is exempt from ethical committee approval because it is not necessary to publish the case report.Not applicable.FD contributed to the conception and design of the study. FD and SLS wrote the manuscript. GE, SC, MR, MDP, MO, and FA contributed to manuscript revisions, read and approved the submitted version.The data are available from the corresponding author upon reasonable request.The authors declare no conflict of interest.


The burden of antimicrobial resistance is a major threat to global health; however, prospective clinical outcome data from Africa are scarce. In Malawi, third-generation cephalosporins are the antibiotics of choice in patients admitted to hospital despite a rapid proliferation of resistance to these drugs. We aimed to quantify the effect of resistance to third-generation cephalosporins on mortality and length of hospital stay among patients with bloodstream infections.Salmonella, and were followed up for 180 days. We characterised blood culture isolates using whole-genome sequencing and used Cox regression models to estimate the effect of resistance to third-generation cephalosporins on length of hospital stay, in-hospital mortality, and survival.We did a prospective cohort study of patients admitted to Queen Elizabeth Central Hospital in Blantyre, Malawi. Patients of all ages who had positive blood cultures for Enterobacterales were included, with the exception of those from the genus Escherichia coli and Klebsiella pneumoniae. Although isolates associated with death were distributed across clades, we identified three E coli clades  that were isolated from 14 patients who all died.Between Jan 31, 2018, and Jan 13, 2020, we recruited 326 patients, from whom 220 (68%) of 326 isolates were resistant to third-generation cephalosporins. The case fatality proportion was 45% (99 of 220) in patients with bloodstream infections that were resistant to third-generation cephalosporins, and 34% (36 of 106) in patients with bloodstream infections that were sensitive to third-generation cephalosporins. Resistance to third-generation cephalosporins was associated with an increased probability of in-hospital mortality , longer hospital stays  and decreased probability of discharge alive . Whole-genome sequencing showed a high diversity of sequence types of both Resistance to third-generation cephalosporins is associated with increased mortality and longer hospital stays in patients with bloodstream infections in Malawi. These data show the urgent need for allocation of resources towards antimicrobial resistance mitigation strategies in Africa.Wellcome Trust and Wellcome Asia and Africa Programme. The burden of antimicrobial resistance is predicted to be high in sub-Saharan Africa, but the sparsity of data linking laboratory results to patient outcomes limits estimates of the burden of disease.5Sentinel surveillance of patients with bacteraemia at Queen Elizabeth Central Hospital (QECH) in Blantyre, Malawi, over the past 25 years has shown a rapid proliferation of resistance to third-generation cephalosporins among Enterobacterales that cause bloodstream infections since the roll-out of ceftriaxone in 2005.7Escherichia coli and Klebsiella pneumoniae isolated from patients with bloodstream infections.To address these data gaps, we aimed to estimate the morbidity and mortality outcomes of patients with bloodstream infections in Blantyre, Malawi, caused by Enterobacterales that are resistant or sensitive to third-generation cephalosporins. Our specific objectives were to estimate the effect of resistance to third-generation cephalosporins on in-hospital mortality, 6-month survival, and length of hospital stay. Through the use of whole-genome sequencing, we also describe the molecular epidemiology of Evidence before this studyWe previously conducted a systematic review of the prevalence and outcomes of bloodstream infections resistant to third-generation cephalosporins in sub-Saharan Africa, searching for papers published between Jan 1, 1990, and Dec 21, 2017, with no language restrictions. By updating the search to March 1, 2021, we added two further studies. We searched PubMed with the search terms“antimicrobial resistance” AND “drug resistance, bacterial” AND “bloodstream” OR “bacteremia” AND a search string that included all sub-Saharan African countries as defined by the UN list of 54 African sovereign states. We found only two prospective studies designed to identify mortality attributed to resistance to third-generation cephalosporins as a primary outcome. Both studies were in children from Senegal and found that bloodstream infection resistant to third-generation cephalosporins remained the only significant independent risk factor for death. However, a 2021 retrospective multisite analysis from South Africa found no effect of resistance to third-generation cephalosporins on patient outcomes. The most comprehensive analysis of the antimicrobial resistance burden worldwide was a modelling study that used all available data. This modelling study estimated that deaths from antimicrobial resistance will be high in sub-Saharan Africa, but identified important data gaps in low-income settings, and no clinical outcome data were available from Malawi.Added value of this studyWe found that outcomes of culture-confirmed Enterobacterales bloodstream infection are unacceptably poor in Malawi, and that resistance to third-generation cephalosporins is associated with increased mortality and longer hospital stays. Using standardised methodology approved by WHO to generate our estimates, we provide the only detailed health outcome data to our knowledge on resistance to one of the most commonly used antibiotics in hospitalised patients in Malawi, and some of the first estimates of the antimicrobial resistance health-care burden in sub-Saharan Africa.Implications of all the available evidenceThe prevalence of resistance to third-generation cephalosporins among bloodstream Enterobacterales in sub-Saharan Africa is high, and access to alternative, WHO Watch and Reserve group antibiotics needed to treat these infections is poor. Bloodstream Enterobacterales are associated with very high mortality irrespective of antimicrobial susceptibility, but resistance to third-generation cephalosporins is associated with increased mortality and longer hospital stays. Future research should combine clinical outcomes with population incidence data for Malawi to generate burden estimates for this country. Ultimately, clinically orientated surveillance that links routinely captured laboratory data to patient outcomes is needed, as are pragmatic trials of the management of sepsis.This prospective, longitudinal, observational cohort study recruited patients from QECH in Blantyre, Malawi. Malawi is a low-income country with low health-care expenditure (approximately 3% of gross domestic product).Salmonella. Patients were excluded if they were unable to consent and had no representative (guardian) available to provide proxy consent, or if they spoke neither English nor Chichewa. For patients who had died by the time their blood culture results were known, a waiver of consent was in place to enable the inclusion of their anonymised data.Salmonella is rare and has not yet been reported in Salmonella enterica serotype Typhi in Malawi.Patients of all ages were eligible for recruitment if their blood culture was positive for bacteria of the order Enterobacterales, with the exception of those from the genus Participants were followed up throughout their stay in hospital until discharge or death, allowing for measurement of in-hospital mortality. To allow for survival analysis and calculation of 6-month mortality, patients or their families were telephoned at 28 days, 3 months, and 6 months after discharge. If a patient had died, family members were asked the date of death.Ethical approval for the study was granted by the University of Malawi College of Medicine Research Ethics Committee (protocol number P.10/17/2299) and by the Liverpool School of Tropical Medicine Research Ethics Committee (protocol number 17-063). The Liverpool School of Tropical Medicine acted as the study sponsor. Written informed consent was obtained from study participants or their guardian if the patient was unable to consent, and from a parent or guardian if the patient was younger than 18 years.Salmonella spp); positive cultures from the weekend were identified on a Monday morning. For patients with positive blood cultures, we collected data on the age and sex of the participants; data on the use of health-care services before the current admission to hospital, including previous hospitalisation, surgery, catheterisation, and antibiotic use; and clinical data including antibiotic treatment during the current hospital admission. The medical records of patients who had died were reviewed and data were captured using the same study case report forms as for participants who were alive. Bloodstream infections were categorised according to their suspected location of onset  and their association with health-care facilities  diagnostic microbiology service provides free blood and cerebrospinal fluid culture testing to patients admitted to the adult and paediatric wards at QECH. Blood cultures were processed at the MLW laboratory, which is quality-assured by the UK National External Quality Assessment Service, and were reviewed daily on weekdays to identify cultures from patients that were positive for Enterobacterales .E coli and K pneumoniae isolates to investigate the presence of hypervirulent lineages. Genomic DNA was extracted from bacterial isolates at MLW , and secondary outcomes were 6-month survival and length of hospital stay (in days).All analyses were carried out in R, version 4.0.2. Sample size considerations are described in the study protocol.The primary outcome of in-hospital mortality was calculated as case fatality proportions, with exact binomial 95% CIs, stratified by third-generation cephalosporin resistance status. For the secondary outcome of 6-month survival, Kaplan-Meier estimates of the survival function over the study period were generated, stratified by third-generation cephalosporin resistance status. Day 0 was the day of infection, which was assumed to be the day of blood-culture sampling. Observations were right-censored at the end of the study follow-up period (180 days). The log-rank test was used to test for differences in survival according to third-generation cephalosporin resistance status.We used Cox regression models to establish the effect of resistance to third-generation cephalosporins on in-hospital mortality. We first hypothesised a causal structure to identify potential confounders in the models, using a directed acyclic graph  to repreIn model 1, we estimated the total effect of resistance to third-generation cephalosporins on death, and in model 2 we included a covariate for whether the administered antibiotic was effective against the bacterial isolate from bloodstream infection. This approach allowed us to control for the confounding effect of antibiotic susceptibility, and therefore estimate the direct effect of resistance to third-generation cephalosporins on death. The approach to variable selection is further described in For the secondary outcome regarding length of hospital stay, Kaplan-Meier curves were generated using the day of infection to the day of discharge from hospital as the timeframe, stratified by susceptibility status, to display differences in the length of stay. To identify the association between resistance to third-generation cephalosporins and length of hospital stay, Cox models were constructed using the day of infection to the day of discharge as the timeframe. Two models were constructed as per the mortality models: the outcome variable was length of hospital stay, and the explanatory variables were health-care exposures and age in model 1, and the same but with the addition of effective antibiotic use in model 2. Observations were right-censored if death occurred before discharge from hospital, and HRs for discharge were presented with 95% CIs.All models were sensitivity-tested for entry point, using the day of hospital admission as the model entry point instead of the day of infection.The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.E coli (identified in 156 [48%] of 326 isolates) and K pneumoniae . 98 (30%) were young infants (<3 months) and, of these, 75 were neonates (<28 days). 169 (52%) of the 326 participants were male and 157 (48%) were female. 89 (57%) of 156 adult participants and 8 (5%) of the 170 children younger than 16 years were living with HIV . HIV pre135 (41%) of the 326 participants died overall. The case fatality proportion was 45% (99 of 220) in patients with infections that were resistant to third-generation cephalosporins, and 34% (36 of 106) in patients with infections that were sensitive to third-generation cephalosporins. Resistance to third-generation cephalosporins was associated with an increased probability of death in hospital in model 1  for participants with bloodstream infections that were sensitive to third-generation cephalosporins and 8·5 days (2–36) for participants with bloodstream infections that were resistant to third-generation cephalosporins. With death considered as a competing risk, resistance to third-generation cephalosporins was associated with decreased probability of hospital discharge in model 1 and in model 2  health-care exposure and hospital-onset bloodstream infections were more frequent in patients with bloodstream infections that were resistant to third-generation cephalosporins than in patients with infections that were sensitive to third-generation cephalosporins  and, excPhenotypic antimicrobial susceptibility patterns were established for all isolates . All isoThe empirical antibiotic regimen contained an antibiotic that was active against the cultured bloodstream isolate (termed an effective antibiotic) in 87 (82%) of 106 bloodstream infections that were sensitive to third-generation cephalosporins but in only 25 (11%) of 220 bloodstream infections that were resistant to third-generation cephalosporins . The medE coli and 66 K pneumoniae genomes passed quality control for bioinformatic analysis. This analysis revealed a high diversity of both E coli and K pneumoniae genomes  were identified, the most common of which was ST131 (44%), followed by ST69 (12%) and ST410 (8%). Among 66 K pneumoniae genomes there were 29 sequence types, including ST14 (12%) and ST35 (12%). The most common gene encoding an extended-spectrum β-lactamase was blaCTX-M-15, both in E coli (present in 51% of genomes) and K pneumoniae (present in 89% of genomes). Other genes encoding extended-spectrum β-lactamases identified in the E coli collection include blaCTX-M-27 (3%) and blaCTX-M-3 (2%). For the K pneumoniae collection, other genes encoding extended-spectrum β-lactamases included blaSHV-106 (24%) and blaOXA-10 (12%). Complete information on sequence type and antimicrobial resistance genes are presented in 119  genomes . Among EE coli and K pneumoniae, although for some large sequence types—such as E coli ST131—isolates branched to form smaller subclades. The within-clade relative frequency of death was higher in some E coli clades than in others, and all patients from whom E coli ST410 (eight patients), ST617 (three patients), and ST648 (three patients) were isolated died. All isolates from these three sequence types had the blaCTX-M-15 gene, but additionally—and unique to the isolates in the clade—ST410 genomes also carried the cephalosporinase blaCMY-2 gene , sequence type, and presence or absence of genes encoding extended-spectrum β-lactamases. The phylogenetic clustering of isolates was consistent with sequence types for both Y-2 gene . No suchWe show that patients with Enterobacterales bloodstream infections in Blantyre, Malawi, have poor clinical outcomes. However, patients with bloodstream infections that were resistant to third-generation cephalosporins had increased risk of death and longer hospital stays than patients with bloodstream infections that were sensitive to third-generation cephalosporins. To our knowledge, our study is the first description of the clinical burden in Malawi of bloodstream infections that are resistant to third-generation cephalosporins, and one of few such prospective studies from sub-Saharan Africa.Ceftriaxone is one of the most frequently used antibiotics for hospitalised patients in Malawi; its broad spectrum of activity, affordability to the health system, and once-daily dosing regimen make it a practical choice, especially where nursing capacity is low. However, ceftriaxone can no longer be the antibiotic of first and last resort in Malawi.The explanation for adverse outcomes for patients with bloodstream infections that are resistant to third-generation cephalosporins is likely to be multifactorial and could include both host and pathogen factors.The prevalence of HIV among adults in the cohort was high, and similar to that in other recent cohorts of patients with infection at QECH.Before our study, it was assumed that the bloodstream infection surveillance data from Blantyre predominantly represented community-acquired infections;E coli and K pneumoniae isolates associated with bloodstream infections and third-generation cephalosporin resistance both in Malawi and worldwide.E coli ST410, which in other settings was found to always produce extended-spectrum β-lactamases and contains a highly carbapenem-resistant clone.The diversity of sequence types represented in our collection are broadly representative of previously described Mycobacterium tuberculosis infection) could worsen outcomes in patients with bloodstream Enterobacterales infections, and these data should ideally be conditioned upon in outcome models. Finally, this study contained insufficient data to include a sepsis severity variable as an adjustment factor in the models, as several of the domains of sepsis scores require parameters that are not available at QECH or require ventilation and inotropic support, which is not routinely assessed.Our study had some limitations. First, recruitment was limited to one hospital owing to the availability of blood-culture facilities. A deeper understanding of the burden of antimicrobial resistance will be developed by including other pathogens and clinical syndromes; larger cohorts to permit age stratification; and routine, clinically orientated surveillance that links laboratory data to patient outcomes.To our knowledge, we describe the first morbidity and mortality burden estimates in Malawi of bloodstream infections that are resistant to third-generation cephalosporins, generated using standardised methodology in line with WHO Global Antimicrobial Resistance and Use Surveillance System recommendations.https://www.ebi.ac.uk/ena) under accession numbers listed in Genome sequence data included in this study are available at the European Nucleotide Archive (NAF received a Wellcome Asia and Africa Programme Grant to the Malawi Liverpool Wellcome Research Programme and a Medical Research Council programme grant. All other authors declare no competing interests.


Lung cancer is the major cause of cancer-related deaths worldwide. The development of targeted therapies has dramatically improved the outcome of lung cancer patients. However, despite an initial response, tumors almost invariably relapse as a result of acquired resistance. In this review, we discuss how lung cancer cells become resistant or tolerant to targeted therapy.Non-small cell lung cancers (NSCLC) harboring activating mutations of the epidermal growth factor receptor (EGFR) are treated with specific tyrosine kinase inhibitors (EGFR-TKIs) of this receptor, resulting in clinically responses that can generally last several months. Unfortunately, EGFR-targeted therapy also favors the emergence of drug tolerant or resistant cells, ultimately resulting in tumor relapse. Recently, cellular barcoding strategies have arisen as a powerful tool to investigate the clonal evolution of these subpopulations in response to anti-cancer drugs. In this review, we provide an overview of the currently available treatment options for NSCLC, focusing on EGFR targeted therapy, and discuss the common mechanisms of resistance to EGFR-TKIs. We also review the characteristics of drug-tolerant persister (DTP) cells and the mechanistic basis of drug tolerance in EGFR-mutant NSCLC. Lastly, we address how cellular barcoding can be applied to investigate the response and the behavior of DTP cells upon EGFR-TKI treatment. Lung cancer is one of the most commonly diagnosed tumors and the leading cause of cancer-related deaths worldwide, claiming an estimated 1.8 million lives annually .NSCLC represents the remaining 85% of lung cancers and includes three major histological subtypes: adenocarcinoma, squamous cell carcinoma, and large cell carcinoma . AdenocaIn the last twenty years, the identification of key genetic events driving tumor growth and survival has dramatically redefined the treatment of NSCLC based on their molecular characteristics . These gEGFR belongs to the ErbB family of receptor tyrosine kinases (RTKs) and binds ligands such as EGF, transforming growth factor alpha (TGF-α) and amphiregulin. The ErbB family consists of four related members, EGFR/ErbB1/HER1, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4, which are implicated in a wide range of biological processes . Under pThe presence of these EGFR-activating mutations has been associated with dramatic responses to treatment with EGFR-TKIs in patients with advanced NSCLC . DiffereThe first generation of EGFR-TKIs is constituted of compounds, such as gefitinib and erlotinib, that reversibly compete with ATP for the binding to the tyrosine kinase pocket of the receptor. These drugs demonstrated improved progression-free survival (PFS), overall survival, and overall response rates (ORR) compared to standard chemotherapy in patients with mutant EGFR NSCLC ,23,24. BPrimary resistance relates to tumors that fail to respond to the treatment. For example, it has been shown that NSCLCs containing certain activating mutations of EGFR, such as exon 20 insertions, and they are insensitive to most EGFR-TKIs . PrimaryAcquired resistance arises in patients after an initial period of drug response, and it can be classified as on-target or off-target. On-target resistance is caused by secondary EGFR mutations that affect the binding of the inhibitor to the receptor A. The moSecond generation inhibitors, including afatinib and dacomitinib, were originally designed in the hope to overcome the T790M-mediated resistance. Unlike gefitinib and erlotinib, these agents are irreversible inhibitors that also target HER2 and HER4 and have shown promising activity against the EGFR-T790M mutation in preclinical models. However, despite encouraging in vitro data, these EGFR-TKIs failed to prevent the emergence of this mutation in patients due to non-selective inhibition of wild-type EGFR ,31. In cGiven the limited efficacy of second-generation EGFR-TKIs in overcoming T790M resistance in NSCLC patients, a number of third generation inhibitors were developed. These agents can form an irreversible covalent bond with the cysteine-797 residue in the ATP binding site of EGFR and showed potent activity against the EGFR-T790M mutation, while poorly inhibiting the wild-type receptor. The first third generation EGFR-TKIs reported was WZ4002, identified by screening of a library of irreversible kinase inhibitor specifically targeting the T790M mutant EGFR. This drug was found 100-fold less potent against wild-type EGFR and 30 to 100 fold more potent against EGFR-T790M . While WDespite its efficacy, resistance to osimertinib inevitably develops. Similar to earlier generations, resistance to third generation EGFR-TKIs is classified as primary and acquired. EGFR-C797S mutation represent the most commonly occurring on-target resistance mechanism on osimertinib treatment ,45. ThisIn order to overcome C797S-mediated acquired resistance, fourth generation EGFR-TKIs were developed. EAI045 is an allosteric, non-ATP competitive inhibitor targeting both the T790M and C797S mutations. It binds to the allosteric sites on EGFR, created by the displacement of the regulatory C-helix in the inactive conformation of the kinase. Due to EGFR dimerization, EAI045 is not effective as a single agent, and it requires the co-administration of the anti-EGFR antibody, cetuximab . RecentlFourth generation EGFR-TKIs appear to be potent and effective against the EGFR-C797S mutations and to have strong antitumor activity in preclinical models. Besides TKIs, other types of inhibitors have been designed that could improve the treatment of patients with EGFR-mutant NSCLC. In particular, the discovery of EGFR degraders holds great promise. A key focus of targeted protein degradation is the development of proteolysis targeting chimeras (PROTACs). PROTACs are heterobifunctional small-molecule degraders, typically consisting of two linked moieties, with one binding the protein of interest and the other binding an E3 ligase. PROTACs recruit the E3 ligase to targeted protein, leading to its selective ubiquitination and degradation by the proteasome ,62. MultDespite the high response rates of EGFR-TKIs, acquired resistance almost invariably occurs. In some cases, this is due to the selection of pre-existing cells harboring well defined genetic resistance mechanisms, such as the T790M mutation or MET amplification, that enable growth during the treatment ,68,69. AThe term of DTP comes from the field of microbiology, where it has been shown that a small fraction of dormant bacteria has the capacity to survive in the presence of antibiotics despite the fact that they do not contain a genetic mechanism of resistance. These drug tolerant cells are able to resume growth and re-establish a drug sensitive population upon drug withdrawal, indicative of a transient and not inheritable resistance mechanism . SimilarSeveral mechanisms have been associated with the ability of drug-tolerant cells to withstand EGFR-TKIs treatment, including chromatin remodeling, activation of bypass pathways, and altered cellular metabolism. In this section, we will summarize the main characteristics of drug-tolerant state described in EGFR-mutant NSCLC. For a more general discussion of how DTP cells can escape cell death in different types of cancer, please refer to our recent review .To gain insight into the underlying molecular mechanism of the drug-tolerant phenotype, Sharma and colleagues analyzed the gene expression profiles of DTP and DTEP and found that these populations are characterized by altered chromatin states. In particular, the authors showed that these cells were able to survive in the presence of EGFR-TKIs by up-regulating the histone demethylase KDM5A, while they could be selectively ablated by the histone deacetylase (HDAC) inhibitor trichostatin A. The study also suggested a role for the insulin-like growth factor 1 receptor (IGF-1R) in the emergence of DTP populations: activation of this receptor was shown to drive drug tolerance by increasing the expression of KDM5A, leading to overall repressive changes in chromatin structure .In line with these findings, Guler et al. showed that EGFR-TKIs induce increased expression of the long-interspersed repeat element 1 (LINE-1), an active retrotransposable element that can propagate and insert randomly throughout the genome, resulting in genome instability. The authors showed that DTPs exhibit a repressive chromatin state by increased methylation of lysines 9 and 27 of histone H3 (H3K9 and H3K27), particularly on the LINE-1 locus, thus decreasing DNA damage in this cell sub-population. Treatment with histone methyltransferase inhibitors, such as tazemetostat, which specifically reduces the global levels of H3K27me3 by inhibiting the activity of enhancer of zeste homolog 2, increases the chromatin accessibility and results in the ablation of DTP cells through derepression of LINE-1 elements .Reactivation of ERK1/2 has been identified as a resistance mechanism to EGFR-TKIs in NSCLC. Ercan and colleagues found that this activation is caused by either an amplification in chromosome 22 harboring the mitogen-activated protein kinase 1 (MAPK1) or by down-regulation of negative regulators of ERK signaling, including the dual specificity phosphatase 6 (DUSP6) . A combiThe tyrosine kinase receptor AXL has been reported to mediate NSCLC tolerance in response to osimertinib treatment. Taniguchi et al. showed that AXL can be activated through suppression of a negative feedback loop involving SPRY4, resulting in AXL heterodimerization with EGFR or HER3. Consistent with these findings, inhibition of AXL could restore the sensitivity to osimertinib and prevent the emergence of DTP cells . Of noteShah and colleagues found that aurora kinase A (AURKA) plays a major role in the emergence of DTP cells induced by EGFR-TKIs treatment. AURKA activation, which is triggered by its regulator TPX2, prevents osimertinib-induced apoptosis by increasing BIM phosphorylation. The authors showed that targeting AURKA and EGFR could reduce the proportion of DTP in vitro and in vivo . Phase IIn another recent study, it was shown that NSCLC cells survive EGFR and MEK dual inhibition by entering a senescence-like state, which is accompanied by up-regulation of the YAP/TEAD pathway. YAP is a transcriptional coactivator that shuttles between the cytoplasm and the nucleus, where it interacts with the transcriptional factor TEAD and regulates the expression of genes that promote cell growth and survival. The authors found that YAP and TEAD cooperate with the EMT transcription factor SLUG to repress the expression of the pro-apoptotic factor BMF, allowing the cells to escape apoptosis and survive. They also developed a new TEAD inhibitor less toxic compared to previous compounds and they showed that it can enhance apoptosis and prevent the emergence of DTP cells in response to EGFR and MEK dual inhibition .The Wnt/β-catenin signaling has also been associated with the maintenance of DTP cells in response to EGFR-TKIs treatment. One study showed that EGFR inhibition results in the activation of β-catenin signaling in a Notch3-dependent manner, leading to the survival of a subpopulation of DTP cells with stem cell-like properties . ConsistRemodeling cellular metabolism, including the ability to maintain the redox balance under nutrient-deprived conditions and other stresses, is one of the hallmarks of cancer . CompareWhile it is becoming more and more evident that acquisition of drug resistance does not rely only on genetic mechanisms, a major question that is still unanswered concerns how tolerant/persister cells originate. Certain cell populations could be enriched because of intrinsic epigenetic properties that favor growth in the presence of the drug through a non-genetic Darwinian selection A. For exAccording to a different model, the tolerant/persister state could be directly induced by the treatment B. This pIt is important to note that Darwinian selection and Lamarckian induction are not mutually exclusive, since certain subpopulations can be selected based on a particular pattern of gene expression, followed by additional changes induced by the drug. For example, the study by Schaffer et al. discussed above identified rare cell subpopulations displaying particular transcriptional profiles that are primed to survive in the presence of the drug. The treatment induces the acquisition of a more stable resistant phenotype through an epigenetic reprogramming, possibly engendered by inhibition of SOX10-mediated differentiation and induction of AP1 and TEAD transcription factors . A betteCellular barcoding is a powerful strategy to investigate the mechanisms of resistance to anti-cancer therapies. The basic principle involves the tagging of individual cells of interest with unique and heritable labels. Different strategies have been developed to integrate the barcodes into the genome of the cells using lentiviral vectors or CRISPR/Cas9 technology.N, where N stands for the number of nucleotides. For example, a 20 bp sequence can yield 420 (~1012) unique barcodes. To avoid the generation of aberrant sequences, such as long repeats or highly unbalanced proportions of G/C or A/T, semi-random pools of DNA barcodes can also be designed, in which certain positions are constrained to one or more specific nucleotides. To ensure that each cell contains only one copy of the vector, and hence one barcode, the lentiviral library is transduced at low multiplicity of infection (MOI), followed by selection of the infected cells, generally using an antibiotic, for which the vector encodes a resistance gene. The barcodes can be “read” by amplifying by PCR the corresponding sequence of the vector from the gDNA of the cells, followed by high-throughput sequencing of the amplicon  systems, the DNA barcode is located in the 3’ UTR of the GFP mRNA. These libraries were used to investigate the fate of reprogrammed fibroblasts  and the Chang and colleagues recently developed a similar approach combining DNA barcoding and scRNA-Seq, called TraCe-seq, to compare the effects of conventional EGFR-TKIs with those of GNE-641, a dual EGFR inhibitor-degrader. They found that GNE-641 was less effective than erlotinib and osimertinib in inhibiting NSCLC cell growth, as well as in reducing the absolute number and the diversity of TraCe-seq barcodes, despite similar levels of MAPK pathway suppression. scRNA-Seq analysis revealed that GNE-641 resistant clones exhibited reduced expression of genes involved in protein processing in the endoplasmic reticulum (ER). The authors showed that the EGFR protein itself plays a crucial role in mediating full cellular efficacy of EGFR-TKIs, as its expression increases ER stress and subsequent pro-death signaling. Consistent with these findings, combination of GNE-641 with low concentrations of ER stress inducers, such as tunicamycin or thapsigargin, strongly enhanced the cytotoxic effects and led to the complete elimination of residual cells. This study uncovered an essential role of the ER protein processing pathway in the response to EGFR targeted therapies .As an alternative strategy of randomly integrating lentiviral libraries, our laboratory developed the CRISPR-barcoding strategy, in which a DNA barcode can be introduced at a specific genome location through CRISPR/Cas9-induced homology directed repair. (HDR). In HDR, a donor DNA co-transfected into the cells functions as a template for precise repair: through appropriate design of the donor DNA, this mechanism can be used to generate a wide range of genetic modifications, including specific point mutations or the insertion of an entire gene. Through CRISPR-barcoding, we inserted a short stretch of degenerated nucleotides in a safe harbor genomic locus of a NSCLC cell line, and we investigated the effects on the clonal architecture induced by EGFR-TKI treatment .The emergence of resistance is a fundamental cancer property, which mostly derives from the fact that individual tumors are composed of an intricate pattern of heterogeneous subclonal populations, functioning as a complex reservoir that fuels the capacity of tumor cells to adapt to environmental conditions. While most EGFR-mutant NSCLCs initially respond to EGFR-TKIs, they ultimately become resistant due to the emergence of small subpopulations of resistant or tolerant cells. Complex barcode libraries have made it possible to trace the behavior of the individual clones that constitute the mass population of cancer cells, thus providing the means to investigate resistance to anti-cancer therapies under a completely different perspective. A better understanding of the underlying causes of drug tolerance and the acquisition of resistance should help improve the efficacy of cancer treatment.


Throughout the COVID-19 pandemic, testing individuals remains a key action. One approach to rapid testing is to consider the olfactory capacities of trained detection dogs.Prospective cohort study in two community COVID-19 screening centers. Two nasopharyngeal swabs (NPS), one saliva and one sweat samples were simultaneously collected. The dog handlers (and the dogs…) were blinded with regards to the Covid status. The diagnostic accuracy of non-invasive detection of SARS-CoV-2 infection by canine olfaction was assessed as compared to nasopharyngeal RT-PCR as the reference standard, saliva RT-PCR and nasopharyngeal antigen testing.335 ambulatory adults (143 symptomatic and 192 asymptomatic) were included. Overall, 109/335 participants tested positive on nasopharyngeal RT-PCR either in symptomatic (78/143) or in asymptomatic participants (31/192). The overall sensitivity of canine detection was 97%  and even reached 100%  in asymptomatic individuals compared to NPS RT-PCR. The specificity was 91% , reaching 94%  for asymptomatic individuals. The sensitivity of canine detection was higher than that of nasopharyngeal antigen testing , but the specificity was lower .Non-invasive detection of SARS-CoV-2 infection by canine olfaction could be one alternative to NPS RT-PCR when it is necessary to obtain a result very quickly according to the same indications as antigenic tests in the context of mass screening. Facing the COVID-19 crisis, early and efficient screenings are needed to limit the spread of the virus, allowing for the prompt isolation of positive individuals, a key action in this constant fight. Presently, most ongoing diagnostic COVID-19 testing involves nasopharyngeal sampling for RT-PCR, nasopharyngeal point-of-care antigen testing or saliva RT-PCR to identify the pathogen. Nasopharyngeal sampling for RT-PCR is the reference test but has the drawback of invasiveness and discomfort  and/or delay (RT-PCR) in obtaining the result.Volatile organic compounds (VOC) have the potential to become a revolutionary and non-invasive approach to medical diagnostic in humans for conditions like cancer and degenerative or infectious diseases. Aksenov , 2 studiOur hypothesis, the object of our previous proof of concept , was basThe objective of this prospective blinded multicenter study was to compare the diagnostic accuracy of the detection of SARS-CoV-2 infection by canine olfaction with the current reference standard  and two alternate diagnostic strategies  in community testing centers.This research is part of the “SALICOV-APHP” study , promoted by Assistance Publique-Hôpitaux de Paris, France. The SALICOV-APHP study was approved the Protection of Persons Committee (CPP) Ile-de France III (number 3840-NI) and is registered on clinicaltrial.gov (NCT04578509). The protocol was approved for the dogs by the committee on the ethics of animal experiments of the Ecole Nationale Vétérinaire d’Alfort. All research procedures were employed in accordance with the relevant guidelines and regulations.Participants were recruited from two COVID screening centers in Paris piloted by APHP. Informed verbal consent was obtained from all subjects. Eligible persons received detailed oral and written information . ParticiSweat samples were collected by dedicated APHP sampling teams by asking the participants to place two sterile surgical compresses under their armpits for 2 min. Samples were stored in sterile medical anti-UV glass containers, disinfected by the sampler’s helper, anonymously coded, then placed into a second plastic envelope. Individual anonymous data were registered by APHP staff for each coded sample. All samples were transferred within the day of collection from the sampling site to the testing site in coolers that were cleaned and disinfected with a 10% aqueous acetone solution after each use. All samples were stored until they were to be sniffed by the dogs in a minus 20°C freezer and were never manipulated without disposable surgical gloves to prevent contamination.Nasopharyngeal samples (NPS) were collected by a trained nurse, and participants were asked to self-collect the saliva sample after swishing saliva in their mouths for 30 seconds .The dogs involved in this study belong to French fire departments  and to the Ministry of the Interior of the United Arab Emirates .They all were trained in the same way according to the training protocol we developed in a previous publication, with line-ups of positive and negative olfaction cones based on positive reinforcement (toys) and validated similarly for their individual sensitivities and specificities . Time toThe welfare of the dogs was fully respected, with toy rewards and a total absence of work-induced physical or mental fatigue. This study was carried out in strict accordance with the recommendations published in the guide for the care and use of animals edited by French law .The testing sessions took place in a dedicated room  in the A™ COVID-19 CE IVD RT PCR Kit . This technique provides results expressed as a cycle threshold (Ct) for each gene target  , and qualitative data as numbers (percentages). The diagnostic accuracy of the index tests was evaluated by calculating sensitivity and specificity. Confidence intervals were calculated by the exact binomial method. Subgroup analyses were performed according to: i) the presence of symptoms on the day of testing, ii) the Ct value of the nasopharyngeal RT-PCR, expressed as low  or high , iii) the consumption of alcohol, coffee, food and smoking or tooth brushing before sample collection and iv) proven previous infection with SARS-CoV-2.Sensitivity analyses were performed considering 6 alternate criteria for positivity for the reference standard: i) ≥ 2 positive targets with nasopharyngeal RT-PCR, ii) ≥ 1 positive target with nasopharyngeal RT-PCR and at least one of the 3 targets with Ct < 32, iii) ≥ 1 positive target with saliva RT-PCR, iv) ≥ 1 positive target with either nasopharyngeal or saliva RT-PCR, v) ≥ 1 positive target with either nasopharyngeal or saliva RT-PCR and at least one of the 3 targets with Ct < 32 and vi) nasopharyngeal antigen test.http://cran.r-project.org/). The reporting of results followed the Standards for Reporting Diagnostic accuracy studies (STARD 2015) guideline [Quantitative variables were compared with Wilcoxon’s test, with a significance level of 5%. The sensitivity and specificity of canine detection and nasopharyngeal antigen tests were compared with McNemar’s test. The statistical analysis was performed using R software  tested positive on nasopharyngeal RT-PCR. The sensitivities and specificities for all the participants and according to the presence/absence of symptoms are presented in Measures of diagnostic accuracy did not differ according to different subgroups: with or without a medical history of COVID, and having received medical treatment or not, tobacco, alcohol or coffee, and gender . In addiAmong the 234 participants who agreed to the nasopharyngeal antigenic test, the sensitivity of canine detection was greater than that of the antigenic test , but the specificity was lower .To assess if the infection with other respiratory viruses could interfere with canine olfaction, saliva samples were tested with a multiplex PCR assay. Remaining saliva samples were available for 283 out of 335 participants. Out of these 283 saliva samples, 87 saliva were positive for SARS-CoV-2 and 25 samples were positive for another pathogen: rhinovirus/enterovirus (n = 11), human coronaviruses (3 OC43 and 2 NL63), parainfluenza 3 (n = 2), bocavirus (n = 1), VRS (n = 2), bocavirus / VRS (n = 1), Influenza A H1 (n = 1), human metapneumovirus (n = 1), Bordetella pertussis (n = 1). Three co-infection with SARS-CoV-2 was identified. Out of 20 individuals tested negative for SARS-CoV-2 in NPS samples but found positive with canine olfaction, 17 samples were tested for multiplex detection in saliva. Fourteen had no other pathogen, two were positive for a coronavirus (NL63 and OC43) and one was positive for SARS-CoV-2. For the diagnosis of viral respiratory infection (other than SARS-CoV-2 infection), the sensitivity of canine detection was 20%  and the specificity was 62% .Our results show the excellent sensitivity of SARS-CoV-2 detection by dogs using nasopharyngeal RT-PCR as the reference for comparison. These results are consistent with the results obtained previously in proof of concepts studies using sweat in hospitalized patients , 8–11.To our knowledge, this study is the first one carried out prospectively in the context of SARS-CoV-2 screening and the first comparing dog detection and antigenic tests.The results detailed showed no real difference in the sensitivity observed in the different subgroups: sensitivity is always over 95% when specificity ranges from 83% to 95%.The results obtained by canine detection using sweat samples are comparable to those of nasopharyngeal antigenic tests. However, canine detection using sweat samples is less invasive than antigenic tests on nasopharyngeal samples. Thus, the detection of SARS-CoV-2 infection by dogs could be an alternative to antigenic tests.Dog screening for SARS-CoV-2 seems specific for COVID-19 infection and not for viral infections in general since only 2 among the 17 saliva samples in false positive patients were positive for a virus other than SARS-CoV-2. Note, however, that in both cases, it was a coronarovirus. We do not have enough patients negative for SARS-CoV-2 and positive for other coronaviruses to know whether the canine detection is specific for SARS-CoV-2 or for all coronaroviruses.Screening people directly, without going through sweat or saliva samples, could also be considered; the advantage would certainly be increased speed. The disadvantage is the fear of dogs exhibited by a significant number of individuals and the risk of dog contamination, which is not possible with the system we have used. Direct detection by dogs should be further evaluated. The Nosaïs team made some proposals to the French ministry of health regarding the use of COVID detection dogs, and these are actually being examined by the High Council for Public Health: autists and trisomic children institutions, Alzheimer patients in nursing homes , airports, harbours, public events, and also schools as it seems very containing for kids to be sampled several times a week.A limitation of non-invasive detection of SARS-CoV-2 infection by canine olfaction is the availability of trained dogs in this approach in light of the very significant needs if canine detection were to be considered as an alternative to antigenic tests. Another limitation is the need for certification of the dogs used for SARS-CoV-2 detection because of the risk of involving dogs whose diagnostic performance is inferior to those shown here. At the time of the study, there was no delta variants detected, but there is no reason to believe that the results would have been different in the presence of the delta variants.To conclude, the results obtained in our prospective study involving 335 individuals who presented voluntarily in one of the APHP testing centers in Paris support the use of canine olfaction as an alternative to antigenic tests. Canine testing is non-invasive and provides immediate and reliable results. As for antigenic tests, positive results must be confirmed by RT-PCR, especially for variant screening. Further studies will be focused on direct sniffing by dogs to evaluate sniffer dogs for mass pre-test in airports, harbors, railways stations, cultural activities or sporting events. Axillary sweat testing could remain useful for small population testing or for mobile units acting on local clusters as an alternative to antigenic tests.S1 Table(DOCX)Click here for additional data file.S2 TableThe others 2 dogs (Oska and Allo) tested only one PCR negative sweat sample each, both detected as negative.(DOCX)Click here for additional data file.