African Americans (AAs) experience greater sleep quality problems than non-Hispanic Whites (NHWs). Meditation may aid in addressing this disparity, although the dosage levels needed to achieve such benefits have not been adequately studied. Smartphone apps present a novel modality for delivering, monitoring, and measuring adherence to meditation protocols.This 6-month dose-response feasibility trial investigated the effects of a breathing awareness meditation (BAM) app, Tension Tamer, on the secondary outcomes of self-reported and actigraphy measures of sleep quality and the modulating effects of ethnicity of AAs and NHWs.A total of 64 prehypertensive adults  were randomized into 3 different Tension Tamer dosage conditions . Sleep quality was assessed at baseline and at 1, 3, and 6 months using the Pittsburgh Sleep Quality Index (PSQI) and 1-week bouts of continuous wrist actigraphy monitoring. The study was conducted between August 2014 and October 2016 (IRB #Pro00020894).P=.03 to P<.001). Longitudinal generalized linear mixed modeling revealed a dose effect modulated by ethnicity (P=.01). Multimethod assessment showed a consistent pattern of NHWs exhibiting the most favorable responses to the 5-min dose; they reported greater improvements in sleep efficiency and quality as well as the PSQI global value than with the 10-min and 15-min doses (range P=.04 to P<.001). Actigraphy findings revealed a consistent, but not statistically significant, pattern in the 5-min group, showing lower fragmentation, longer sleep duration, and higher efficiency than the other 2 dosage conditions. Among AAs, actigraphy indicated lower sleep fragmentation with the 5-min dose compared with the 10-min and 15-min doses . The 10-min dose showed longer sleep duration than the 5-min and 15-min doses . The 5-min dose also exhibited significantly longer average sleep than the 15-min dose (P=.03).At baseline, PSQI and actigraphy data indicated that AAs had shorter sleep duration, greater sleep disturbance, poorer efficiency, and worse quality of sleep (range These findings indicate the need for further study of the potential modulating influence of ethnicity on the impact of BAM on sleep indices and user-centered exploration to ascertain the potential merits of refining the Tension Tamer app with attention to cultural tailoring among AAs and NHWs with pre-existing sleep complaints. Obtaining an adequate amount and quality of sleep is essential for optimal health. Chronically insufficient sleep negatively impacts daytime function and has been linked to an increased risk of hypertension, stroke, cardiovascular disease, obesity, stress, and poor mood -6. One oMitigating stress through positive cognitive behavioral coping strategies is a common treatment approach to improve sleep quality . MeditatAlthough the benefits of meditation on stress are positive, sleep outcomes have been mixed. Neuendorf et al  reviewedLarge-scale dissemination of meditation programs using traditional modes of training may be challenging. Individual or group-based in-person training and monitoring methodologies have typically been used ,17; howeTechnology-delivered meditation programs are an innovative option to overcome several of these barriers. Unfortunately, few programs have been rigorously evaluated. In particular, meditation programs delivered by smartphone apps are widely available  and can Research has also consistently shown greater self-reported sleep quality problems  among African Americans (AAs) than non-Hispanic Whites (NHWs) ,27-31. TFurther studies are warranted to determine if meditation techniques, such as BAM, have a beneficial dose-response influence upon indices of sleep based on self-reported and objective verification via accelerometry. A recently completed 6-month dose-response study using Tension Tamer, a BAM smartphone app, provides this opportunity. The primary goal of the feasibility trial was to determine the potential dose-response impact on resting BP among prehypertensive adults . We examThis paper reports on the exploratory analysis of 3 different doses of BAM on sleep quality changes in adult NHWs and AAs with prehypertension. We hypothesized that increased doses of BAM would impart greater improvements in self-reported and actigraphy-derived measures of sleep quality. We also hypothesized that AAs would exhibit poorer baseline levels of sleep quality compared with NHWs.A total of 287 prehypertensive adults were recruited using paper advertising and word-of-mouth referrals at an academic health center in southeastern United States. Study candidates were contacted by phone to initiate eligibility screening and were scheduled for the first of 3 clinic BP evaluations. Inclusion criteria included participants aged between 18 and 90 years; being NHWs or AAs; and having systolic prehypertension based on the Seventh Report of the National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (120 mm Hg-139 mm Hg) on 3 consecutive visits, each at least 2 days apart. Exclusion criteria included unwillingness to accept randomization into 1 of 3 dosage groups, current medication regimens that affected BP, inability to open and navigate the Tension Tamer smartphone app after a hands-on training, or inability of participants to position their fingers over their smartphone camera because of tremors. Additional exclusion criteria included being currently pregnant, lactating, or having intention to become pregnant during the trial, participating in another research study of any kind, inability to read or speak English, and not having smartphone signal at the participant’s place of residence. The study was conducted between August, 2014 and October, 2016.Tension Tamer captures real-time heart rate (HR) with a proprietary software involving reflective photoplethysmography by using the smartphone video camera and light-emitting diode (LED) flashlight . The vidUsers have the option to turn off (or reactivate) the audio BAM instructions, set session midway and ending alerts (chime or gong), and select different background themes for screenshots . All research procedures were approved by the institutional review board before conducting the study (IRB #Pro00020894).Complete details of the study protocol have been reported elsewhere . After ibelly breaths. At the start of each session, a chime indicated the start with an on-screen timer. Additional chimes notified the user of the midpoint and the end of the session. Numeric feedback was immediately displayed on a 4-beat rolling average basis throughout the session. A graph was displayed immediately following the session, showing the average HR response during each minute of the session  or the Android operating system on each participant’s personal smartphone. If the subject did not have a smartphone, an Apple iPhone 4 or 5 or Samsung Galaxy 4 or 5 was provided at no cost for the duration of the study. A staff member trained in the use of the Tension Tamer app was present to provide guidance as needed during the introductory tutorial. The Tension Tamer app tutorial included Tension Tamer videos explaining BAM, demonstrating diaphragmatic breathing, and explaining how to operate the app’s features, including obtaining video camera HR capture using the fingertip. An audio recording of a British female voice guided them through their first BAM session. Participants were instructed to place the tip of a finger over the camera’s lens and LED light. The app would vibrate if the finger were misaligned and prompt for realignment. Participants were directed in the audio guide to sit comfortably, close their eyes, and attend to the movement of their diaphragm while taking deep, slow, shown in . The traAn ActiGraph monitor was then fitted on their wrist, and participants were given instructions to wear the device for 7 consecutive days and to complete a daily sleep log. The same procedure was followed at each subsequent laboratory evaluation at 1, 3, and 6 months. Participants were compensated US $145 for their time after completion of each evaluation.Actigraphy was captured using the ActiSleep monitor . The ActiSleep monitor is a research-grade accelerometer that provides objective estimates of physical movement and includes multiple sleep parameters that have been validated against polysomnography -54. The PSQI, administered at each evaluation, is a well-validated self-report questionnaire of sleep quality over the previous month ,56-59. TIn addition to the objective physical activity data provided by the ActiSleep monitor, a single-item self-report question was completed at each evaluation for comparison. The question, derived from the Sweat Index , was “HoAdherence was measured using the encrypted time and date stamped HR data obtained via the Tension Tamer app’s built-in photoplethysmogram that were automatically relayed to our institution’s server infrastructure. These data provided objective measures of adherence . Adherence was reported as a monthly percentage calculated as the average of daily scores  across the number of days in the month.P=.05. Analyses of longitudinal dose intervention effects from baseline to final 6-month evaluation for self-reported (PSQI) and actigraphy sleep indices were performed on the intention-to-treat (ITT) sample comprising all participants who completed at least four Tension Tamer sessions following completion of the baseline evaluation (n=57). A generalized linear mixed (GLM) model approach was used to account for the correlation of repeated measurements within patients [Demographic variables were analyzed using the analysis of variance for continuous data and chi-square tests for categorical data. For all analyses, statistical significance was set at patients ,65. The The second level of GLM modeling included ethnicity as a fixed effect in addition to the previously included effects because of the statistically significant differences in most actigraphy-derived sleep indices observed at baseline. We estimated changes in sleep for each subject over the trial  and the within-subject longitudinal trajectories  and summarized the mean longitudinal trajectory within each dose group.All sleep outcome variables were examined for normality using the Shapiro-Wilk test. With 18 subjects randomized to each intervention group, we had 80% power to detect at least a 0.96 SD unit effect size among the 3 groups  for sleep outcomes. SAS Statistical Software version 9.4 (SAS Institute Inc) and SPSS Statistics version 25.0 (IBM Corp) were used to perform the analyses.The study was approved in July 2014 (IRB#Pro00020894) and was performed between August, 2014 and October, 2016. A total of 287 potential candidates were screened . OverallP=.07 or greater). In addition, baseline sleep characteristics were not significantly different among dosage groups for any self-reported or objective variable .Overall, 47% (27/57) of the participants were female and 49% (28/57) were African American. As illustrated in P=.04 to P=.001). Importantly, ethnicity differences were observed in 6 of the 7 baseline sleep quality indices , with 50% (14/28) falling above the clinical cutoff level for sleep disturbance  [P=.02 to P=.001).On the basis of the PSQI, AAs self-reported significantly poorer sleep efficiency  and poorer sleep quality ; core >5) ,61 compaP=.08 or greater). Among the ActiGraph-derived variables, a significant dose effect was observed only for sleep fragmentation (P=.03). Collapsed across the 3 evaluation time points, the 5-min dose condition exhibited the lowest sleep fragmentation  compared with both the 10-min  and 15-min  dose conditions.Longitudinal GLM models evaluating the impact of Tension Tamer dosage conditions on sleep variables at 1, 3, and 6 months, controlling for baseline levels and adherence, did not reveal any statistically significant dose×time interaction terms in any of the PSQI-based sleep outcomes  and on the PSQI sleep efficiency factor (P=.03).GLM modeling was conducted with ethnicity as a covariate in the model . EthniciP=.05; duration P=.03). The sleep measure was the dependent variable, whereas dosage was the primary independent variable adjusted for visit, dose-by-visit interaction term, baseline level of particular outcome measure, and adherence.As a result of the above findings, an ethnicity×dose interaction term was added to the model to determine whether ethnicity modulated the effects of dosage level on sleep outcomes. Significant interaction effects were observed for 2 of the 3 actigraphic measures (efficiency P=.02). Similarly, they also reported better scores for the sleep quality factor compared with the 10-min and 15-min dose conditions, with the difference for the 15-min condition being statistically significant (P=.006). Finally, the 5-min condition reported significantly better global sleep quality compared with both the 10-min and 15-min conditions .The NHWs exhibited a consistent pattern across both self-reported and ActiGraph-derived measures. The 5-min dose resulted in the greatest improvements in sleep measures compared with the other 2 dosage conditions. On the basis of the PSQI data, participants in the 5-min condition reported significantly better values for the sleep efficiency factor compared with both the 10-min and 15-min conditions  and 15-min groups; however, the latter difference did not reach statistical significance. Similarly, the 5-min condition exhibited lower fragmentation and longer sleep duration compared with the other 2 conditions, but the differences were not statistically significant.The ActiGraph findings supported these patterns among NHWs, with the 5-min group showing higher sleep efficiency compared with the 10-min . The 10-min condition showed the highest sleep duration, which was significantly longer than both the 5-min and 15-min conditions . The 5-min condition also exhibited significantly longer sleep duration compared with the 15-min condition (P=.03).Among AAs, the actigraphy findings indicated that the 5-min condition exhibited significantly lower sleep fragmentation than both the 10-min and 15-min conditions (P=.08). Furthermore, the overall pattern of PSQI factor scores did not emulate the actigraphy findings. For example, the 15-min dose condition, which had the shortest actigraphy-derived sleep duration and greatest fragmentation during sleep, had the best sleep quality factor, global sleep quality scores, and daily disturbance factor score.With regard to the self-reported results among AAs, none of the PSQI factor scores were significantly and differentially impacted by the Tension Tamer dosage, except for PSQI daily disturbances. The 15-min group showed a trend of lower daily disturbances compared with the 10-min group  and better global sleep quality (P=.05) than AAs. The 15-min dose condition revealed a different pattern in which AAs reported better scores in sleep quality and daily disturbance factors and lower global sleep quality scores than NHWs; however, none of these differences reached statistical significance .Per the PSQI outcomes, NHWs reported a consistent pattern of greater improvement than AAs for each PSQI factor  and global sleep quality for the 5-min and 10-min dose conditions. However, the only statistically significant differences observed were for the 5-min dose condition, in which NHWs reported greater sleep efficiency (P=.02), lower fragmentation (P=.02), and longer sleep duration (P=.002) than AAs.The ActiGraph results revealed a pattern in which NHWs exhibited comparable or better sleep efficiency and fragmentation than AAs across all 3 doses. They also exhibited longer sleep durations at the 5-min and 15-min dose conditions. However, statistically significant differences were observed in the 15-min dose condition only. NHWs exhibited better efficiency  compared with NHWs (r range 0.45 to 0.56) ,67-69.On the other hand, among the NHWs, a consistent pattern across both self-reported and ActiGraph-derived indices indicated that the 5-min dose resulted in the greatest improvements in sleep measures. On the basis of the PSQI, the 5-min condition reported significantly greater improvements in sleep efficiency compared with both the 10-min and 15-min conditions. Similarly, the 5-min condition reported better sleep quality compared with the other 2 conditions, with the difference for the 15-min condition being statistically significant. Finally, the 5-min dose condition reported significantly better PSQI global scores  compared with both 10-min and 15-min dose conditions. The pattern of actigraphy findings supported the self-reported findings. The 5-min condition exhibited higher sleep efficiency scores compared with both 10-min and 15-min conditions, with the 10-min comparison being statistically significant. Similarly, the 5-min condition had lower fragmentation compared with the other 2 dosage conditions; however, the differences did not reach statistical significance.Evaluations of potential ethnicity differences in response to the 3 Tension Tamer doses revealed a consistent pattern of significantly better responsivity among the NHWs. ActiGraph-derived measures indicated significantly greater improvements among NHWs in sleep efficiency, reduced fragmentation, and longer sleep duration compared with AAs at each dosage condition. The PSQI findings provided support to the actigraphy findings, with NHWs reporting a pattern of higher quality, efficiency, and better PSQI global sleep scores for both 5-min and 10-min dose conditions compared with AAs. Compared with NHWs, AAs in the 15-min dose condition reported better sleep quality and PSQI daily disturbance scores, with a trend toward better global sleep scores. A higher use of Tension Tamer in the 5-min group across the trial may have contributed to the generally greater beneficial influence on sleep quality. Anecdotally, the 5-min participants appeared to more often note at posttrial key informant interviews using Tension Tamer immediately before going to bed, which may have played a role as well.As noted earlier, the findings have been mixed with regard to the impact of meditation on sleep ,13,17,24Use of cellular technology provides the possibility of disseminating efficacious mind-body intervention programs such as BAM to larger audiences, circumventing barriers such as travel, childcare, and other expense-related issues. In addition, the penetration of smartphone-assisted technology into lower income, rural, and urban areas enables these programs to be widely disseminated and available. This is increasingly important as 81% of adults own a smartphone as of June 2019 . Our exiA limitation of this study was that the Tension Tamer app was not designed to specifically target individuals with sleep issues but was rather for those with prehypertension. As a result, we did not include eligibility criteria related to sleep problems, insomnia, or collect sleep medication usage. Therefore, bias toward the null hypothesis may be an explanation for some of the findings. In future studies, inclusion of targeted intervention materials in addressing sleep-related barriers in prehypertensive patients may have a greater effect. Such tactics could include increasing basic sleep hygiene awareness or using the Tension Tamer app in combination with cognitive behavioral therapy . FurtherThis study was also not designed to examine the daily timing of BAM sessions around bedtime routines. A properly powered trial using a refined Tension Tamer app, including BAM as part of bedtime preparation rituals, is needed to understand how the timing and dosage of BAM or other meditation techniques may provide the most beneficial effect on sleep outcomes. We also did not investigate any related mechanistic evaluations such as electromyography or other common sleep tests such as polysomnography , which cTo our knowledge, this is the only study that has examined the influence of varying doses of meditation delivered via a smartphone app on multimethod-derived indices of sleep  in a multiethnic population not diagnosed with sleep disturbance issues. A high level of methodological rigor was incorporated, including random assignment to dosage condition, automated feedback of dosage adherence, and postsession HR graphs to provide reinforcement for engaging with the Tension Tamer app. Ethnicity was found to play a modulating role on the effects of Tension Tamer. Formative evaluation adapted from the development of Tension Tamer  can targ


In December 2019, an outbreak of pneumonia cases in Wuhan, China was attributed to a novel coronavirus that was eventually recognized as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently identified as coronavirus disease 2019 (COVID-19), it has been declared a pandemic by the World Health Organization given its rapid global transmission. Various cardiovascular complications have been reported, including heart failure, myocarditis, acute coronary syndrome and arrhythmias, both atrial and ventricular. Regarding arrhythmias, onset from time of infection is variable but usually ranges from several days to a week. We hereby present a case of a COVID-19 positive patient presenting with new onset atrial fibrillation. In December 2019, a cluster of cases of pneumonia of unknown cause were identified in Wuhan, Hubei, China that was eventually attributed to a novel enveloped coronavirus, currently named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) . SeveralA 37-year-old male patient with no significant past medical history presented to the emergency department with left foot pain of one-day duration following direct trauma. No other symptoms were reported on presentation, including palpitations, chest pain, shortness of breath or dizziness. Of importance, the patient reports multiple family members testing positive for SARS-CoV-2 two weeks ago as well as himself having experienced mild flu-like symptoms several days ago. Regarding social history, the patient denied any current or prior tobacco smoking, alcohol consumption or other drug abuse. Initial vital signs were notable for heart rate of 90 beats per minute, blood pressure of 152/89 mmHg, respiratory rate of 20 breaths per minute and an oxygen saturation of 100% on room air. The patient's weight is 86 kilograms with a calculated body mass index of 26.8. A 12-lead electrocardiogram demonstrated an irregularly irregular rhythm consistent with atrial fibrillation  score for atrial fibrillation was zero, and therefore anticoagulation was not indicated. The patient was eventually discharged home with a new diagnosis of atrial fibrillation. Despite it being difficult to establish a causation between COVID-19 and atrial fibrillation in our patient, especially in the absence of an echocardiogram, this report aims to highlight the possible association between those two conditions.Cardiac arrhythmias have been reported in several recent reports in the literature in relation to hospitalized patients with COVID-19. Additionally, it was demonstrated to be associated with a higher risk of mortality . HoweverRegarding pathophysiology, multiple mechanisms exist in which COVID-19 can trigger cardiac arrhythmias. Potential contributing factors include metabolic derangements, acidosis and hypoxia . MoreoveIt is noteworthy to mention that malignant cardiac arrhythmias, such as sustained ventricular tachycardia and ventricular fibrillation, have been observed in hospitalized patients with COVID-19. Despite the limited clinical and published data, this most likely occurs in the setting of COVID-19-associated acute cardiac injury and myocarditis . MoreoveIn light of the emerging pandemic of COVID-19, several cardiovascular complications including arrhythmias are gaining worldwide recognition. Awareness of such entities and their theoretical pathophysiology is required for early recognition. Future research is needed to further investigate those observations and the associated clinical outcomes.


Pre-eclampsia is an elusive condition to diagnose and a complex disease to manage. There have been recent developments in prediction, prevention, diagnosis, and management. Risk modelling has been used to identify women at highest risk of developing pre-eclampsia as well as predicting maternal adverse outcomes in confirmed disease. New evidence has shown that aspirin prophylaxis significantly reduces early onset pre-eclampsia as well as preterm birth. The criteria for the diagnosis of pre-eclampsia are evolving, and proteinuria is no longer a pre-requisite to make a diagnosis. Angiogenic biomarker testing accelerates diagnosis as well as minimises adverse maternal outcomes and has been incorporated into national guidelines. Emerging evidence demonstrates that expedited delivery in late preterm pre-eclampsia may be protective against maternal adverse outcomes but increase the risk of neonatal unit admission. Both women and their offspring are at increased risk of long-term health complications following pre-eclampsia, and it is important that postnatal health is optimised. This article summarises recent developments in the field of pre-eclampsia research, evaluating the impact on clinical care for women at risk of, or with suspected or confirmed, pre-eclampsia. Pregnancies complicated by pre-eclampsia show an increase in maternal and perinatal morbidity and mortality. The International Society for the Study of Hypertension in Pregnancy published updated guidance on the diagnosis and management of hypertensive disorders of pregnancy in 2018. The revised definition of pre-eclampsia is de novo hypertension after 20 weeks’ gestation with one or more of proteinuria, maternal organ dysfunction , or foetal growth restriction3. The classification of hypertensive disorders is shown in 4.Hypertensive disorders of pregnancy complicate 10% of all pregnancies and are estimated to cause 40,000 maternal deaths worldwide each year5. Therefore, risk prediction has been a substantial focus within the field of pre-eclampsia research.Pre-eclampsia is notoriously difficult to predict. Accurate prediction models identifying women at high risk of disease would enable targeted prophylaxis with aspirin as well as enhanced surveillance for high-risk women to mitigate adverse outcomes. Inadequate recognition of risk contributes to substandard care associated with maternal deaths6. Women should take aspirin if they have one strong risk factor or more than one moderate risk factor for pre-eclampsia. These risk factors were highlighted by a large meta-analysis of clinical risk factors for pre-eclampsia, which analysed over 25 million pregnancies7. Additional risk factors have also been identified and are listed in Table 28. Inherited susceptibility may also play a part, and a large genome-wide association study in the offspring of 4,380 cases of pre-eclampsia identified a susceptibility locus near the FLT1 gene encoding Fms-like tyrosine kinase 19.The updated National Institute for Health and Care Excellence (NICE) guidance on hypertension in pregnancy recommends a list of risk factors to identify high-risk women who should be advised to take aspirin 75–150 mg daily from 12 weeks’ gestation until birth13; placental protein 1314; angiopoietin 1 and 215; inhibin A and Activin A; soluble endoglin and soluble fms-like tyrosine kinase-1 (sFlt-1)16; and human chorionic gonadotropin (hCG)17.Pre-eclampsia is challenging to predict. There have been many studies investigating multiple-marker algorithms to predict pre-eclampsia in a similar way to first-trimester aneuploidy screening. It has been demonstrated that there are significant differences in first-trimester levels of pregnancy-associated plasma protein A (PAPP-A), a disintegrin and metalloproteinase 12 (ADAM12), and placental growth factor (PlGF)18. A model using parity, history of pre-eclampsia, ethnicity, chronic hypertension, and conception method achieved an area under the curve (AUC) of 0.76  to predict early onset pre-eclampsia, which represents a modest test. Nine studies comparing simple versus specialised models in the same population reported AUCs favouring specialised models. A simple model achieved fewer false positives than a guideline-recommended risk factor list such as the NICE hypertension in pregnancy guideline, but the clinical value of different models to guide aspirin prophylaxis still needs to be determined.A large systematic review compared “simple” risk models for pre-eclampsia that use routinely collected maternal characteristics against “specialised” models that include specialised tests19. The authors have previously demonstrated that their triple test is superior to risk factor-based screening and can predict 90% of early pre-eclampsia (necessitating delivery before 32 weeks’ gestation) and 75% of preterm pre-eclampsia at a screen-positive rate of 10%20. Using this two-stage strategy of screening would mean that only 70% of the population would require the triple test whilst achieving similar detection and screen-positive rates. This would clearly have financial benefits over intensive screening of the whole population and may be a promising area of development.Wright and colleagues recently studied the effect of two-stage screening, with a first stage screening of the whole population based on risk factors and a second stage “triple test”  for those identified as high risk21. Aspirin prophylaxis has recently been investigated in the ASPRE trial, which was a multicentre double-blind randomised controlled trial including 1,620 women (aspirin versus placebo in pregnancies at high risk for preterm pre-eclampsia)22. This trial demonstrated that 150 mg aspirin resulted in a 60% reduction in preterm pre-eclampsia  and a 90% reduction in early onset pre-eclampsia, with no significant effect on term pre-eclampsia. The authors hypothesise that the larger effect size seen in their trial may be due to the higher dose of aspirin taken at night and commenced prior to 16 weeks’ gestation. Similar results were found in a recent meta-analysis23. Recent evidence has also emerged from the ASPIRIN study, a randomised, double-blind, placebo-controlled trial of low-dose aspirin for the prevention of preterm birth in 11,976 women from low-income countries24. This demonstrated a small but significant reduction in preterm birth  and a larger reduction in preterm birth before 34 weeks’ gestation in women with hypertensive disorders . Therefore, aspirin is a safe and simple strategy that is likely to have far-reaching benefit. However, non-adherence may be an underestimated problem. A study of 220 women found that 44% of women were non-adherent and that these women had significantly higher rates of early onset pre-eclampsia , late-onset pre-eclampsia , and intrauterine growth restriction 25. Furthermore, self-reported adherence does not represent actual adherence.Accurate prediction of pre-eclampsia will facilitate targeted prophylaxis with aspirin. Initial studies demonstrated that early administration of prophylactic aspirin in high-risk women prior to 16 weeks’ gestation reduced the risk of pre-eclampsia by 17%, with an 8% relative risk reduction of preterm birth and a 14% reduction in foetal and neonatal death26, but robust evidence from randomised controlled trials is lacking. A Cochrane systematic review found that high-dose calcium supplementation during pregnancy reduces the risk of pre-eclampsia and preterm birth, especially in women with a diet deficient in calcium (<600 mg/day)27. Calcium supplementation is not recommended in women with normal dietary calcium intake, but the World Health Organization recommends daily calcium supplementation (1.5–2 g) for pregnant women in populations with low dietary calcium intake. Supplementation with vitamins C and E has no benefit in preventing pre-eclampsia and is not recommended28.Other interventions have been investigated to assess impact on pre-eclampsia, including nutritional supplements and dietary and lifestyle modifications. Some studies have suggested benefit with vitamin D supplementationde novo hypertension after 20 weeks’ gestation with one or more of proteinuria, maternal organ dysfunction , or foetal growth restriction. Hypertension is classified as a systolic blood pressure of 140 mmHg or higher and/or a diastolic blood pressure of 90 mmHg or higher at or after 20 weeks’ gestation3. Approximately 25% of those with a background of chronic hypertension will develop super-imposed pre-eclampsia, which is defined as a worsening of hypertension in association with new-onset maternal organ dysfunction3.Pre-eclampsia is elusive to diagnose. Pre-eclampsia is 29. OPTIMUM  is a randomised controlled trial of blood pressure self-monitoring during pregnancy for women with chronic hypertension, which aims to assess the feasibility and most appropriate outcome measures for a larger trial30. The BUMP trial (blood pressure monitoring in high-risk pregnancy to improve the detection and monitoring of hypertension) is a randomised controlled trial to determine whether self-monitoring leads to earlier diagnosis of raised blood pressure and lower mean systolic blood pressure between baseline and delivery31. Recruitment has finished for both trials and the results are awaited.Recent research has focused on whether ambulatory or home blood pressure monitoring impacts maternal outcomes in pre-eclampsia. A systematic review from 2002 concluded that there was no randomised controlled trial evidence to support ambulatory blood pressure monitoring and recommended adequately powered randomised trials to evaluate this32. The DAPPA study (diagnostic accuracy in pre-eclampsia using proteinuria assessment) recruited 959 women, of whom 417 had severe pre-eclampsia, and compared spot protein:creatinine ratio (PCR) and spot albumin:creatinine ratio (ACR) against the reference standard of 24 hour urine collection33. They found that the diagnostic accuracy of PCR and ACR was similar to a 24-hour urine collection and that ACR had a significantly higher sensitivity of 99% compared to 90% with PCR. Therefore, NICE recommends dipstick screening for proteinuria and, if positive (1+ or more), then ACR or PCR should be used to quantify proteinuria6.Additionally, assessment of proteinuria is variable. The gold standard for assessment of proteinuria was previously a 24-hour urine collection. However, this was logistically challenging and prone to error34. There is a need for better methods of diagnosis and risk stratification of women at risk of pre-eclampsia. Angiogenic biomarkers are closely linked to the pathophysiology of pre-eclampsia, and abnormalities in angiogenic biomarker concentrations such as PlGF and sFlt-1 have been identified up to 10 weeks before the clinical onset of disease35. The role of angiogenic biomarkers in diagnosis and risk stratification in suspected pre-eclampsia has been investigated, and there have been recent developments.The diagnosis of pre-eclampsia is complex, particularly on a background of medical co-morbidities. Hypertension, proteinuria, and biochemical and haematological abnormalities are tertiary, downstream features of established disease, which may be absent even in women presenting with eclampsia36. Low PlGF concentrations demonstrated high sensitivity  and negative predictive value  for diagnosing pre-eclampsia necessitating delivery within 14 days in women with suspected pre-eclampsia before 35 weeks’ gestation . Prediction of short-term outcome in pregnant women with suspected pre-eclampsia (PROGNOSIS) was a prospective, multicentre observational study investigating the clinical utility of a sFlt-1:PlGF ratio, with 500 women included in a development cohort to determine a ratio cut-off and 550 women as a validation cohort37. sFlt-1:PlGF ratios of 38 or lower have a high negative predictive value  and an 80% sensitivity (95% CI 51.9–95.7) for diagnosing pre-eclampsia within 1 week. The positive predictive value of an sFlt-1:PlGF ratio above 38 for a diagnosis of pre-eclampsia within 4 weeks was 36.7% (95% CI 28.4–45.7), and sensitivity was 66.2% (95% CI 54.0–77.0). The authors conclude that a high negative predictive value is crucial in the evaluation of suspected pre-eclampsia, as failure to detect imminent disease could have important consequences for the woman or foetus.Chappell and colleagues investigated the diagnostic accuracy of PlGF in the prospective, multicentre PELICAN study, which included 625 womenestation 36. PlGF 38. This found that a low PlGF concentration of <100 pg/ml was significantly associated with preterm delivery as well as adverse neonatal outcomes  and maternal outcomes 39. The authors conclude that PlGF may be useful for risk stratification for women presenting with suspected pre-eclampsia. The PARROT trial  was a stepped-wedge cluster-randomised controlled trial of revealed versus concealed PlGF testing implemented alongside a clinical management algorithm40. This trial enrolled 1,023 women and demonstrated that time to diagnosis was reduced from 4.1 to 1.9 days, and severe adverse maternal outcomes were reduced from 5.4 to 3.8% . There was no difference in gestational age at delivery or adverse perinatal outcomes. Finally, the INSPIRE trial  evaluated the use of the sFlt-1:PlGF ratio42. This trial found no difference in the primary endpoint of hospitalisation within 24 hours of testing but demonstrated that a higher proportion of women were admitted who subsequently developed pre-eclampsia over the following 7 days, demonstrating more appropriate use of resources.More recently, the PlGF test and sFlt-1:PlGF ratio have been assessed in further studies. The PETRA study (pre-eclampsia triage by rapid assay) was a large prospective cohort study in North America44. In a secondary cost-saving analysis of the PARROT trial, PlGF testing resulted in a total cost-saving of £149 per woman (based on £70 per PlGF test)45. This more conservative estimate is likely because of an appropriate redistribution of resources rather than an overall reduction in resources as anticipated by hypothetical analyses. In view of the evidence for clinical and cost benefit, the updated NICE Guideline on Hypertension in Pregnancy recommends a single PlGF-based test at the time of presentation with suspected preterm pre-eclampsia between 20 and 34+6 weeks’ gestation46.Initial economic models predicted that PlGF-based testing may afford a cost-saving of between £330 and £1,032 per woman testedP <0.0001)47. A case-control study of 11 cases of foetal death and 829 controls found that an angiogenic index-1 value <2.5th centile was associated with a 29-fold increase in the risk of foetal death48. Another study of 314 pregnant women with suspected small-for-gestational-age foetuses  found that angiogenic biomarkers could identify the majority of women who subsequently developed pre-eclampsia or indicated preterm delivery, with AUC greater than 80%49. However, in other studies, there have been important false negatives, including stillbirth50.Unfavourable angiogenic biomarker profiles are particularly linked to adverse perinatal outcomes, including foetal death and severe intrauterine growth restriction. In a study of 412 women with suspected pre-eclampsia, women with pre-eclampsia and adverse outcomes had lower PlGF and higher sFlt-1:PlGF ratio than women without adverse outcomes  or alanine aminotransaminase (ALT). The PREP-S prediction model is intended for use up to 34 weeks’ gestation and provides robust estimates of the overall risk of adverse maternal outcomes52. The PREP-S model requires maternal age, gestational age, medical co-morbidities, PCR, urea, creatinine, ALT, platelets, systolic blood pressure, pulse oximetry, presence of exaggerated tendon reflexes, and treatment with anti-hypertensive drugs or magnesium sulphate. However, neither of these models predict adverse perinatal outcomes.There are now externally validated risk prediction models available to predict adverse maternal outcomes once pre-eclampsia has been diagnosed and to guide clinical management, including timing of delivery, antenatal steroids, magnesium sulphate, and transfer to high-level care. The fullPIERS model is intended for use at any time in pregnancy and predicts adverse outcomes in the next 48 hours6. This is an important change from previous practice, when treatment was recommended if blood pressure exceeded 150/100 mmHg and reflects evidence from CHIPS (Control of Hypertension in Pregnancy Study)53. This trial was an international multicentre randomised controlled trial comparing “tight” blood pressure control (target diastolic blood pressure 85 mmHg) to “less tight” blood pressure control (target diastolic blood pressure 100 mmHg) in women with non-severe non-proteinuric maternal hypertension. A total of 981 women were randomised, and the results demonstrated that those with “tight” control were less likely to experience severe maternal hypertension (P <0.001), without any effect on adverse perinatal outcome or birthweight <10th centile. Severe maternal hypertension was significantly associated with the primary composite outcome of perinatal loss or high-level neonatal care for >48 hours as well as serious maternal complications. As this study included women with non-proteinuric hypertension, the results should be extrapolated to pre-eclampsia with caution, but there may be benefit in tighter control of blood pressure.The revised NICE Guideline recommends offering treatment for hypertension in pregnancy if systolic blood pressure is sustained above 140 mmHg or diastolic blood pressure is sustained above 90 mmHg. Once anti-hypertensive treatment has been started, the target blood pressure is 135/85 mmHg6. It is vital that women are provided with information on the benefits of treatment and the side effects of the various options for treatment to enable shared decision making and informed choice.The revised NICE guidance recommends labetalol as the first-line treatment for hypertension in pregnancy, with nifedipine recommended if labetalol is not suitable and methyldopa recommended if neither labetalol or nifedipine are suitable or tolerated54. Recent research has been investigating the optimum time to deliver between 34 and 37 weeks’ gestation to prevent morbidity for women and their babies.Both NICE in the United Kingdom and the American College of Obstetricians and Gynecologists recommend delivery at 37 weeks’ gestation for women with confirmed pre-eclampsia. Before 34 weeks’ gestation, expectant management is advised, as iatrogenic preterm delivery before 34 weeks’ gestation is associated with worse neonatal adverse outcomes 55. A total of 703 women were enrolled from 51 hospitals in the Netherlands. The results demonstrated that immediate delivery (by induction or elective caesarean section) was associated with a non-significant reduction in a composite of severe maternal adverse outcomes . However, there was an increased risk of neonatal respiratory distress syndrome in the immediate delivery group , and thus the authors conclude that routine expedited delivery does not seem to be justified. Two-year infant follow up from the HYPITAT-II trial showed a significant increase in neurodevelopmental delay, but this was not evident at 5-year follow up57.The HYPITAT-II randomised controlled trial (immediate delivery versus expectant monitoring for hypertensive disorders of pregnancy between 34 and 37 weeks of gestation) investigated the effect of immediate delivery compared to expectant monitoring on maternal and neonatal outcomes in women with hypertensive disorders in late preterm pregnancy+6 weeks’ gestation58. This trial of 901 women demonstrated that planned delivery was associated with a significant reduction in adverse maternal outcomes  and a significant increase in neonatal unit admissions for prematurity but without any other indicators of neonatal morbidity. The authors conclude that this trade-off should be discussed with women with late-preterm pre-eclampsia to enable informed shared decision making. This larger trial suggests maternal benefit in earlier delivery from 34 weeks in all women with pre-eclampsia when combined with the previous data demonstrating beneficial trends. A meta-analysis is under way. Evidence from a low-income setting is required where both potential harms and benefits are considerably greater and where the vast burden of pre-eclampsia disease is found.The PHOENIX randomised controlled trial  investigated planned delivery versus expectant management in women diagnosed with late preterm pre-eclampsia between 34 and 3659. Numerous high-quality studies have now firmly established that pre-eclampsia increases lifetime risk of cardiovascular disease. A meta-analysis of over 3 million women demonstrated increased risk of vascular disease, with a RR of 3.7 for hypertension and 2.16 for ischaemic heart disease60. This effect is multiplied after recurrent pre-eclampsia, with a hazard ratio of 2.04 for all-cause mortality, 5.10 for stroke, and 3.30 for ischaemic heart disease in a retrospective cohort study of 57,384 women61. Follow up from the HYPITAT trial showed that almost half of women with early onset pre-eclampsia developed hypertension over 2–5 years post-delivery compared to 39% and 25% of women in the pregnancy-induced hypertension and late-onset pre-eclampsia groups, respectively62. Long-term follow up from PHOENIX has also been undertaken and is pending.A study of 31 women with a history of early onset pre-eclampsia found subclinical impairment of left ventricular function 12 years after pre-eclampsia compared to women with late-onset pre-eclampsia or normotensive pregnancies (n = 62)63. As well as links with long-term cardiovascular health, pre-eclampsia has also been associated with peripartum cardiomyopathy, and the latter has similarly been associated with an imbalance of angiogenic factors64.Pre-eclampsia has long-term health implications for womenin utero exposure to pre-eclampsia resulted in 5.17 mmHg greater systolic blood pressure and 4.06 mmHg greater diastolic blood pressure in childhood65. Similar results were demonstrated in a 20-year prospective follow-up cohort of 2,868 young adults, which found that exposure to hypertension in utero increased the risk of hypertension in offspring by 2.5 times66.Long-term risks impact not only women but also their offspring. A meta-analysis of 53,029 individuals demonstrated that 6. Angiogenic biomarkers accelerate diagnosis and minimise adverse maternal outcomes when used in the assessment of suspected disease. Their use will enable risk stratification and appropriate resource redistribution and are similarly recommended in updated national guidelines. There is evidence regarding timing of delivery between 34 and 37 weeks’ gestation in confirmed pre-eclampsia, and shared decision-making with women regarding anti-hypertensive medication and timing of delivery is vital.Important evidence is still emerging to improve the diagnosis and management of this complex disease. Screening models using specialised tests show promise, and aspirin may have far-reaching effects in reducing both early onset pre-eclampsia and preterm birth. New evidence is awaited regarding the impact of home blood pressure monitoring on severe hypertension and adverse outcomes. The CHIPS trial demonstrated better outcomes with tight blood pressure control in pregnancy hypertension, and this has been incorporated into national guidance67. It is a triumph of modern obstetrics that there is fewer than one maternal death from hypertensive disorders of pregnancy per million births. Optimising management to improve perinatal outcomes remains a challenge, particularly in a global setting.The most devastating complication in pre-eclampsia is maternal death. The MBRACE report  found that hypertension-related maternal deaths are at the lowest rate everACR, albumin:creatinine ratio; ALT, alanine aminotransaminase; AUC, area under the curve; CHIPS, Control of Hypertension in Pregnancy Study; CI, confidence interval; NICE, National Institute for Health and Care Excellence; OR, odds ratio; PCR, protein:creatinine ratio; PlGF, placental growth factor; RR, relative risk; sFlt-1, soluble fms-like tyrosine kinase 1.


Frankliniella occidentalis, the western flower thrips. We conducted thrips performance and preference experiments combined with plant gene expression, phytohormone and total free amino acid analyses to determine if systemically-infected tomato plants modulate primary metabolic and defense-related pathways to culminate into a more favorable environment for the vector. In a greenhouse setting, we documented a significant increase in the number of offspring produced by F. occidentalis on TSWV-infected tomato plants compared to mock-inoculated plants, and in choice test assays, females exhibited enhanced settling on TSWV-infected leaves. Microarray analysis combined with phytohormone signaling pathway analysis revealed reciprocal modulation of key phytohormone pathways under dual attack, possibly indicating a coordinated and dampening defense against the vector on infected plants. TSWV infection, alone or in combination with thrips, suppressed genes associated with photosynthesis and chloroplast function thereby significantly impacting primary metabolism of the host plant, and hierarchical cluster and network analyses revealed that many of these genes were co-regulated with phytohormone defense signaling genes. TSWV infection increased expression of genes related to protein synthesis and degradation which was reflected in the increased total free amino acid content in virus-infected plants that harbored higher thrips populations. These results suggest coordinated gene networks that regulate plant primary metabolism and defense responses rendering virus-infected plants more conducive for vector colonization, an outcome that is potentially beneficial to the vector and the virus when considered within the context of the complex transmission biology of TSWV. To our knowledge this is the first study to identify global transcriptional networks that underlie the TSWV-thrips interaction as compared to a single mechanistic approach. Findings of this study increase our fundamental knowledge of host plant-virus-vector interactions and identifies underlying mechanisms of induced host susceptibility to the insect vector.Several plant viruses modulate vector fitness and behavior in ways that may enhance virus transmission. Previous studies have documented indirect, plant-mediated effects of tomato spotted wilt virus (TSWV) infection on the fecundity, growth and survival of its principal thrips vector,  Most plant-pathogenic viruses depend exclusively on insects for transmission to plants . It woulTomato spotted wilt orthotospovirus  is ranked among the top 10 most economically important plant viruses worldwide   . The thrergande) . The virergande) . After iergande)  but therergande)  and longergande)  on infecergande)  and neutergande)  effects Arabidopsis thaliana and Nicotiana benthamiana. Arabidopsis mutants. The authors suggested that increased performance of thrips on TSWV-infected plants was caused by a reduction of the JA-regulated plant defense, which was suppressed by an increase in SA-regulated plant defense responses in virus-infected plants. More recently, F. occidentalis by directly interacting with MYCs, key regulators of the JA signaling pathway. To date, there have been four studies on transcriptional responses (microarray and RNA-Seq) to TSWV in host plants which report changes in plant immune defenses and metabolism in virus-infected plants  in defense responses to pathogens and insects is well-established . HoweverF. occidentalis, on the plant host, tomato, Solanum lycopersicum L. First, we performed replicated greenhouse and laboratory experiments to confirm that TSWV altered vector performance and behavior on TSWV-infected plants. To characterize plant molecular mechanisms, microarray analyses using the Affymetrix Tomato GeneChip® were performed to analyze gene expression in tomato plants receiving one of four treatments: (i) mock-inoculated, or (ii) systemically-infected with TSWV, and subsequently infested (iii) with or (iv) without non-viruliferous thrips. The microarray represents myriad genes associated with primary metabolism, including chloroplast function, cell wall modification, protein synthesis, as well as defense- and stress-related genes associated with phytohormone signaling pathways . In addition, we analyzed phytohormone levels and total free amino acid content in plants exposed to single and dual challengers. This is the first comprehensive analysis of plant-mediated mechanisms (genes and metabolites) that potentially improve the quality of TSWV-infected plants for its thrips vectors.In the current study, we sought to elucidate the plant-mediated mechanisms underlying the interaction between TSWV and its insect vector, s infestation alone; (3) TSWV infection and thrips infestation, and (4) mock-inoculated, healthy controls. Each treatment was replicated three or four times  in a randomized complete block design with one plant per treatment per block. The experiment was conducted three times .The greenhouse experiment consisted of four treatments: (1) TSWV infection alone; (2) thripTomato spotted wilt virus (isolate TSWV-MT2) was maintained by mechanical inoculations on caged tomato plants (S. lycopersicum cv Moneymaker) under greenhouse conditions as per (Phaseolus vulgaris) as described by s as per . A colon® potting soil and housed in one thrips-proof-screened  cage in a greenhouse room. Plants were fertilized once a week with Miracle Gro®-Water Soluble All Purpose Plant Food (24-8-16) NPK. The temperatures in the greenhouse ranged from 23 to 25°C and the photoperiod was 16:8 h (L:D).Tomato plants (cv Moneymaker) were grown in 6-inch pots filled with Metro mixTo generate TSWV-infected plants, 3-week-old plants were mechanically-inoculated with TSWV from infected plant tissue or mock-inoculated with buffer and healthy plant tissue. Virus inoculum was prepared by grinding two to three young symptomatic tomato leaves in ice-cold 5–10 ml of inoculation buffer (10 mM sodium sulfite and 5% wt/vol celite) using a pre-chilled mortar and pestle. Inoculum was applied and dragged lightly with a cotton swab over the surface of all fully-expanded leaves on the plant. Approximately 2 weeks after inoculation, plants were visually inspected for TSWV symptoms, i.e., stunting and deformation, chlorotic ring spots, mosaic patterns, and leaf-bronzing, and the most uniform group of symptomatic plants were chosen for each experiment.TM  and sealed with silicone. The top of the container was covered by thrips-proof-screen secured by rubber bands. These single-plant cages prevented cross-contamination between treatments. Cohorts of 40 adult females from the laboratory colony, 7-day post eclosion were transferred into 1.5 ml microcentrifuge tubes and then allowed to escape in a synchronous manner from one tube placed at the base of each plant.Individual 5-week-old symptomatic and mock-inoculated control plants were moved to single-plant cages constructed from 19-liter cardboard ice cream buckets  with four 14 cm × 27 cm apertures cut into the side walls. The four apertures were covered with No-Thrips Insect ScreenThrips feeding leaf damage index (LDI) was determined using a modified visual rating scale according to Thrips performance on virus-infected plants or mock-inoculated plants was defined as the number of offspring that emerged from leaf tissue at 7-day post release of adult thrips. For each biological replicate, 2-sample rank tests  using Minitab v.14  was performed to determine if the median count of offspring obtained from TSWV-infected plants was similar to that obtained from healthy, mock-inoculated plants. Data from all three experiments were pooled together and a two-way analysis of variance in Minitab using a GLM with treatment and biological replicate as fixed effects.To determine adult thrips settling preference, assays using detached-leaflets were conducted as per . Same-agr = 0.739, P < 0.0001). One leaflet was processed for gene expression analysis  and the other was processed to determine phytohormone contents. Leaflet samples were flash-frozen in liquid nitrogen and stored at −80°C. To obtain enough leaf tissue (approximately 5 g) required for determination of total free amino acid content, a third leaf immediately basal to the leaflets chosen for microarray and phytohormone analyses was simultaneously harvested and freeze-dried.Preliminary observations revealed that thrips preferred to feed on older  leaves, so leaflets were harvested from 10th–11th youngest leaf, counting down from the top to the base of the plant. From each of the three experiments , two same-age, paired leaflets (on each side of leaf rachis) were harvested. We harvested leaflets exhibiting similar leaf damage ratings  between the two thrips treatments  to reduce the confounding effect of variation due to amount of feeding . In the ® at Kansas State University, Integrated Genomics Center. Each GeneChip® contains more than 10,000 probe sets for over 9,200 genes with each gene being represented by at least one probe set containing 25-mer oligonucleotides. Hybridization intensities of scanned microarrays for each of the three biological replicates were generated with Gene Chip® Operating System, GCOS (Affymetrix Inc.). Global scaling was applied for each GeneChip to adjust the Target Intensity (TGT) Value to an arbitrary target of 500 so that hybridization intensity of all chips was equivalent. In addition, expressed genes were identified by GCOS, using a detection algorithm and assigned a present, marginal, or absent call for genes represented by each probe set on the array . Microarray data files (.CEL) were analyzed using GeneSpring 10.1  and normalized using RMA (Robust Multichip Average) algorithm. Differentially-expressed genes were selected using 2 criteria: (i) an expression ratio of at least ±2-fold change and (ii) P ≤ 0.05 in ANOVA tests comparing log2  of treatment to the mock control. Differentially-expressed genes were assigned functional annotations with Blast2GO software . The colors range from red to green, showing the expression of genes in each treatment group ranging from highly similar (green) to less similar (red). The heatmap was plotted from the adjacency matrix of the network, using the R heatmap  function. The Pearson correlation coefficients depicts correlation between genes and treatments based on the average fold change values from 3 replicates for each treatment.® were obtained manually based on literature and from Blast2GO biological processes1 . The weighted gene correlation network analysis method WGCNA,  can be u4  was used4 (5  was adapytoscape  functionselected . As our selected . The resleEF1) as the internal reference gene for normalization because expression of this gene was found to be invariant with TSWV infection or herbivore challenge in the microarray experiments and had been previously shown to be stably-expressed in Moneymaker tomato systemically-infected with tobacco rattle virus  and OPR3, AOS and CI (JA pathway), and RNA-directed RNA polymerase 1 (RDR1). We chose elongation factor 1-alpha  genes and ΔCt is the difference in Ct-values  between a treatment and mock control. The average Ct value (n = 3) obtained for the mock control for each target and reference gene was determined and used in the RER calculations. To estimate virus titer, the normalized abundance of TSWV N or NSs RNA (genomic and transcript RNA) was calculated using the Pfaffl inverse equation (ErefCtref/ENCtN as described previously (The relative abundance of target RNA was determined for each virus and herbivore treatment compared to the mock control. The relative expression ratio (RER) equation  was calcequation : ErefCtreviously .10-transformed phytohormone contents  in Minitab using a GLM that included treatment and biological replicate as two fixed factors and their interaction term as the random factor. The analyses revealed no apparent main effect or treatment interaction for any of the phytohormones measured due to biological replicate; therefore, data was combined for the three biological replicates and one-way ANOVA was performed to determine the main effect of treatment on phytohormone concentration. Pairwise treatment comparisons were performed using Tukey’s family error rate (P ≤ 0.05).Frozen tomato leaflet samples from each biological replicate were sent to The Donald Danforth Proteomics & Mass Spectrometry Facility, St. Louis, MO, United States for chemical extraction and quantification of SA, JA, ABA, jasmonyl-isoleucine (JA-Ile), and 12-oxo-phytodienoic acid (OPDA) using liquid chromatography–electrospray tandem mass spectrometry as per . Analysig for 5 min. The dry residue was dissolved in 2.5 ml of 0.1 N HCl and kept at −20°C until assayed. Colorimetric procedures were adapted to Technicon Autoanalyzer II for simultaneous determination of total free amino acid content based on an internal standard (leucine) in plant tissue samples [modified from Frozen tomato leaf samples obtained from each experiment were sent to the Ruminant Nutrition Laboratory at Kansas State University for extraction and quantification of total free amino acids. Total free amino acid content was analyzed using a modified protocol described . BrieflyF = 11.78, df = 1, P = 0.003)  and the interaction between number of offspring and biological replication  on thrips population. On average, there were twofold more thrips offspring on virus-infected plants compared to mock-inoculated plants . Leaf damage caused by thrips was quantified and no apparent differences were detected between thrips on healthy or virus-infected plants  associated with TSWV-infected leaflets compared to mock-inoculated leaflets .The effect of TSWV infection of tomato plants on thrips performance (number of offspring) was evaluated 7-days after adult females were released onto individual plants. Our data revealed significant differences in the number of offspring (first and second instar larvae) produced on virus-infected plants compared to mock-inoculated plants (= 0.003) . There wleaflets . This trP < 0.05, regardless of magnitude of fold change). Of these sequences, 307, 171, and 424 genes were significantly expressed by at least twofold and P ≤ 0.05 in TSWV, thrips, and TSWV + thrips challenged plants compared to the mock-inoculated controls  represented on the Tomato GeneChip, 1,722 sequences were differentially-expressed in plants challenged by the various treatments compared to mock-inoculated, healthy plants  terms into biological process, cellular component and molecular function with relevance to plant health and responses. Overall, a greater proportion of genes responded to TSWV infection alone and combined treatment compared to thrips feeding alone in all three GO categories . The GO-We examined the microarray hybridization data for possible interaction between SA and JA pathways. The magnitude and direction of differential expression of all known phytohormone genes in the Tomato GeneChip are shown in We analyzed other phytohormones that are known to interact with SA and JA/ET, namely ABA and auxin and that were differentially-expressed in our microarray analysis. Virus infection alone did not significantly impact ABA-related genes (66% showed similar expression to mock) and 33% ABA-related genes were down-regulated . Thrips Genes involved in photosynthesis were largely down-regulated in all three treatments . Virus iVirus infection altered gene expression in both upward and downward direction. Among the up-regulated genes (29%), cell wall degradation genes such as expansin, some cellulases and beta-1, 4-glucanases that are also involved in the SA-pathway . In contA majority of genes involved in protein synthesis such as constituents of 40S, 50S, and 60S ribosomal subunits and genes associated with protein degradation such as those that encode 26S proteasome and involved in ubiquitination were induced in response to TSWV and the combination treatment  of individual genes in the treatment relative to the control  between normalized abundance of N RNA and NSs RNA. Analysis of variance revealed significantly lower virus titers in leaves from TSWV-infected plants infested with F. occidentalis compared to plants infected with TSWV alone , indicating that thrips infestation on infected leaves for 1 week significantly influenced virus accumulation. There were significant correlations (n = 6) between TSWV titer and Log2RER for NPR1 , and Cathepsin D (CI) inhibitor protein , an indication that expression of SA- and JA-associated transcripts may be quantitatively associated with the extent of TSWV infection  . We inclWV alone  , we quantified the levels of SA, JA, JA-Ile, and OPDA in leaf tissues 7-days post thrips release. Same-age leaflets that were immediately basal (older) to the leaves harvested for microarray analysis were used for phytohormone analysis. TSWV infection alone or in combination with thrips enhanced SA content in tomato leaves . Thrips F = 10.87, df = 2, P = 0.0002) and treatment  but not the interaction term  on the total free amino acid content. Overall, TSWV infection alone and in combination with thrips feeding harbored greater total free amino acid content  compared to mock-inoculated and thrips –fed plants   (ctively) .F. occidentalis. While previous research identified plant defense response as a key molecular mechanism through which TSWV affects its vector  between the treatments (data not shown). Taken together, we hypothesize that increased aggregation and population growth of thrips on virus-infected plants can potentially increase the number of viruliferous vectors, which in turn could be expected to increase virus secondary spread by thrips dispersal to neighboring plants. While it is well documented that TSWV spread in various vegetable crops grown in Australia   and ordeOPR3, LOX AND AOS in TSWV-infected plants. In the current study, we used mechanical leaf-rub inoculation to infect plants with TSWV and thrips were released 2-weeks post virus-inoculation, by which time we expected that wound-related responses to be attenuated. Wounding and insect feeding activate JA-regulated wound response genes such as proteinase inhibitors . They too found that genes such as rubisco and Chaperonin and others involved in the photosynthesis pathway were largely suppressed in response to TSWV infection.Although at different time points post-infection, similar trends documented in the transcriptome profiles of tomato and findings . We collfindings . Moreovefindings . Catoni Sw-7) and susceptible line challenged with TSWV infection, the authors found cell wall-related genes to be down-regulated in the resistant line, Sw-7 and only three out of the five pectinesterases were up-regulated (PR-1 and PR-5 (osmotin) that modulate callose and lignin deposition in the cell wall leading to restricted virus movement in the resistant line. Indeed, our study also found increased expression of PR-1 and PR-5 in virus-infected plants. These findings provide evidence supporting the dual regulatory role of cell wall organization genes in plant structure and defense. It is possible that suppression of cell wall genes renders the plant more susceptible to the penetration of mouthparts of thrips vectors.In this study, we found that virus infection altered genes related to cell wall organization or biogenesis. Most cell-wall-modification enzymes such as xyloglucan endochitinase, endotransglycosylase/hydrolases and pectinesterase expression levels were decreased in virus-infected plants. In contrast, cell wall degradation genes such as expansin, some cellulases and beta-1, 3-glucanases were upregulated. In agreement with our findings, egulated . The autlht1 (lysine histidine transporter 1) mutant of Arabidopsis has significantly reduced contents of glutamine, alanine, and proline in comparison with wild-type plants and showed enhanced resistance to diverse bacterial, fungal, and oomycete pathogens  prior to release of female thrips on these plants. Interestingly, we found that thrips – on infected plants for only 1 week prior to sampling – had a negative effect on virus titer as measured by real time qRT-PCR using two viral genes, TSWV-N and NSs. The expression of canonical virus-activated pathways was similar for TSWV alone and the dual treatment leading us to hypothesize that (i) the dual attack may further compromise plant health and thus creates a less suitable host, or (ii) the presence of the insect vector alters the virus composition in the plant host through a yet unidentified pathway or mechanism. Further exploration of this phenomenon is warranted and it will be interesting to determine if thrips perception by the plant alters the virus in a way to promote acquisition similar to transmission morphs described for non-circulative viruses .The datasets generated for this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/PN, DM, JN, AW, and DR conceived and designed the experiments. PN and DR performed the experiments. PN, DR, and JC analyzed the data. PN, DM, JN, AW, and DR contributed to reagents, materials, and analysis tools. PN and DR wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This randomized clinical trial uses data from the Early Venous Reflux Ablation trial to evaluate the long-term clinical and cost benefits of combined early endovenous ablation and compression compared with compression therapy alone and deferred ablation for treating superficial venous reflux of the leg. In patients with venous leg ulceration and superficial reflux, what is the clinical and cost-effectiveness of early endovenous ablation of reflux?In this 450-patient, multicenter, randomized clinical trial, early endovenous ablation with compression accelerated venous ulcer healing, reduced the overall incidence of ulcer recurrence, and was highly cost-effective compared with compression with deferred intervention.To deliver clinical and cost benefits, leg ulcer care pathways should be revised to include early assessment and treatment of superficial venous reflux. One-year outcomes from the Early Venous Reflux Ablation (EVRA) randomized trial showed accelerated venous leg ulcer healing and greater ulcer-free time for participants who are treated with early endovenous ablation of lower extremity superficial reflux.To evaluate the clinical and cost-effectiveness of early endovenous ablation of superficial venous reflux in patients with venous leg ulceration.Between October 24, 2013, and September 27, 2016, the EVRA randomized clinical trial enrolled 450 participants (450 legs) with venous leg ulceration of less than 6 months’ duration and superficial venous reflux. Initially, 6555 patients were assessed for eligibility, and 6105 were excluded for reasons including ulcer duration greater than 6 months, healed ulcer by the time of randomization, deep venous occlusive disease, and insufficient superficial venous reflux to warrant ablation therapy, among others. A total of 426 of 450 participants (94.7%) from the vascular surgery departments of 20 hospitals in the United Kingdom were included in the analysis for ulcer recurrence. Surgeons, participants, and follow-up assessors were not blinded to the treatment group. Data were analyzed from August 11 to November 4, 2019.Patients were randomly assigned to receive compression therapy with early endovenous ablation within 2 weeks of randomization  or compression with deferred endovenous treatment of superficial venous reflux . Endovenous modality and strategy were left to the preference of the treating clinical team.The primary outcome for the extended phase was time to first ulcer recurrence. Secondary outcomes included ulcer recurrence rate and cost-effectiveness.P = .28). Ulcers recurred at a lower rate of 0.11 per person-year in the early-intervention group compared with 0.16 per person-year in the deferred-intervention group . Time to ulcer healing was shorter in the early-intervention group for primary ulcers . At 3 years, early intervention was 91.6% likely to be cost-effective at a willingness to pay of £20 000 ($26 283) per quality-adjusted life year and 90.8% likely at a threshold of £35 000 ($45 995) per quality-adjusted life year.The early-intervention group consisted of 224 participants . The deferred-intervention group consisted of 226 participants . Of the 426 participants whose leg ulcer had healed, 121 (28.4%) experienced at least 1 recurrence during follow-up. There was no clear difference in time to first ulcer recurrence between the 2 groups (hazard ratio, 0.82; 95% CI, 0.57-1.17; Early endovenous ablation of superficial venous reflux was highly likely to be cost-effective over a 3-year horizon compared with deferred intervention. Early intervention accelerated the healing of venous leg ulcers and reduced the overall incidence of ulcer recurrence.ISRCTN02335796ClinicalTrials.gov identifier:  Superficial venous surgery has largely been superseded by endovenous ablation procedures , but long-term outcomes in patients with venous leg ulcers are unknown.Venous leg ulceration is the most extreme manifestation of chronic venous disease, and worldwide prevalence is increasing.Extended follow-up was performed for participants in the EVRA trial to evaluate the influence of early endovenous ablation of superficial venous reflux on outcomes up to 5 years for participants with venous leg ulcers.This parallel-group randomized controlled trial was conducted in 20 centers in the United Kingdom  reporting guideline.The study design and 1-year outcomes of the EVRA trial have been published previously.Participants were assigned randomly in a 1:1 ratio to receive compression therapy and endovenous ablation within 2 weeks (early-intervention group) or to receive compression therapy alone with deferred endovenous ablation once the ulcer had healed, or after 6 months if the ulcer had not healed (deferred-intervention group). Randomization sequences were created in advance for each center by a trial statistician, and randomly permuted blocks were used with 2 block sizes. Surgeons, participants, and follow-up assessors were not blinded to the treatment group. Photographic verification for healing of primary ulceration was performed by clinical experts blinded to treatment allocation.Wound care and compression therapy were guided by local protocols, and multilayer elastic compression , short-stretch bandaging, and compression hosiery were all accepted. The endovenous treatment was left to the discretion of the responsible clinical teams, with endovenous thermal ablation modalities (laser or radiofrequency ablation), ultrasound-guided foam sclerotherapy, or nonthermal nontumescent endovenous interventions performed alone or in combination. Decisions regarding treatment of branch varicosities or perforators were left to physician choice. EVRA trial centers had extensive experience in performing endovenous ablation procedures. Participants in the early-intervention group underwent follow-up duplex ultrasound assessment 6 weeks after endovenous ablation, and additional interventions for superficial venous reflux in either group were performed at the discretion of the treating clinical teams. All participants were advised to use compression hosiery after ulcer healing, guided by local policy; additional duplex ultrasound assessment was not in the study protocol.Telephone follow-up for all living participants was performed between October 2018 and March 2019 to obtain primary and secondary end point data. Where possible, participants in the EVRA trial were reminded at the 12-month visit to record any recurrent ulcers and health care visits in a participant diary to aid in subsequent recall. In the extended-phase follow-up, participants were asked on the telephone (using a standardized questionnaire) about ulcer recurrences (defined as any wound on the study leg) and asked to recall dates of recurrence, subsequent healing, and details of additional treatments. Hospital and community clinical records were reviewed for further verification, and further calls were made to participants to clarify discrepancies.A disease-specific quality-of-life assessment (Aberdeen Varicose Vein Questionnaire) and 2 generic quality-of-life assessments   leg with no scab or requirement for dressings, and a blinded verification process was used to confirm healing.7The secondary outcome measures were time to first ulcer recurrence from date of randomization, the proportion of participants with recurrent ulceration at different time points (ulcer recurrence rate), time to healing of index and recurrent ulcers, length of time free from ulcers from randomization to final follow-up (ulcer-free time), recurrent ulcer incidence rate and incidence rate ratio, participant-reported health-related quality of life, and cost-effectiveness.Statistical analyses of the data were performed from August 11, 2019, to November 4, 2019. The trial was designed to detect a 15% absolute difference in ulcer-healing rates at 24 weeks  with 90% power and 2-sided alpha level of 5%. Assuming 90% of the participants in the EVRA trial would achieve ulcer healing and 15% losses to follow-up, death, or withdrawal from the study, we estimated that 344 participants would be available for analysis of ulcer recurrence. For extended follow-up analysis, we calculated that this was sufficient to detect a 15% difference in ulcer recurrence (30% in the early-intervention group vs 45% in the deferred-intervention group) with 82% power or a 20% difference in ulcer recurrence (30% in the early-intervention group and 50% in the deferred-intervention group) with 97% power.8 Moreover, the incidence rate of recurrent ulcers (ulcers per person-years) and incidence rate ratios with 95% CIs were calculated. Ulcer-free time was defined as the total number of days that the reference leg remained healed during the entire follow-up period. We used a Cox regression model adjusted for center, patient age, ulcer size, and ulcer chronicity, as mentioned, as well as length of follow-up (as a fixed effect) to test the hypothesis that there was no difference in ulcer-free time between the early-intervention and deferred-intervention groups. Participants who did not consent to the extended follow-up are included to 12 months only. Adverse events were recorded.The null hypothesis was that there is no difference in time to ulcer recurrence between the early-intervention group and the deferred-intervention group. This was tested using Cox regression with center as a random effect and participant age, ulcer size, and chronicity as fixed effects. We used Cox regression, adjusted as mentioned, to test for differences in time to healing of primary ulcer and recurrent ulcers. Ulcer recurrence rates (unadjusted) were calculated at annual time points up to 4 years with 95% CIs using the Kaplan-Meier method.3 We used 3-level mixed models to assess differences in each quality-of-life measure between the 2 treatment groups. All analyses were performed on intention-to-treat. Participants whose primary ulcer did not heal were not eligible for analysis for ulcer recurrence, but were included in all other secondary analyses. There were no statistical adjustments for multiple testing. We performed per-protocol analyses for time to ulcer healing and time to first ulcer recurrence, and statistical significance was set at 5%.Differences between study groups to 1 year for each quality-of-life measure have been published previously.5 The price year was the 2017 to 2018 period. Discounting was applied according to UK Government guidelines .9 Study conduct and reporting complied with current guidelines for economic evaluation.10 We collected details of resource use in hospital and community care related to venous leg ulcer treatment, adverse events, or complications of venous leg ulcers or treatments. We used case note review and questionnaires completed at baseline and monthly thereafter to 1 year, plus 1 further telephone follow-up between October 2018 and March 2019, with notes review for additional verification. Each item of resource use was multiplied by unit costs obtained from published literature,11 national unit costs,13 and manufacturers’ list prices to calculate overall costs for each participant  from the perspective of the UK National Health Service and Personal Social Services over a 3-year time horizon. Results to 1 year have been published previously.14 Cost and EQ-5D-5L data were analyzed using mixed models and total mean costs, and total mean QALYs were estimated for the 3-year time horizon. Sensitivity analyses used an alternative tariff for the EQ-5D-5L, per-protocol analysis and 4- and 5-year time horizons.15 Uncertainty in mean costs and QALYs was quantified using bootstrapping and presented using cost-effectiveness acceptability curves  underwent endovenous ablation within 2 weeks of randomization. Of 226 participants in the deferred-intervention group, 171 (75.6%) were treated with endovenous ablation within 12 months  or deferred intervention (226 participants) in addition to compression therapy. The early-intervention group consisted of 224 participants . The deferred-intervention group consisted of 226 participants  .3,4 Of 22 months . The finData were collected over the telephone and from medical notes or from medical notes alone for 399 of 422 participants (94.5%) still participating at 1 year . Median Of the 426 participants whose leg ulcer had healed, 121 (28.4%) experienced at least 1 recurrence. There were 175 episodes of recurrent ulceration during follow-up .Time to first recurrence from ulcer healing  was similar in the early-intervention group and the deferred-intervention group  (P = .43) .P = .28) A. CalculP = .002) .Time to ulcer healing of the primary ulcer was shorter in the early-intervention group compared with the deferred-intervention group  B. UnlikeThe median ulcer-free time was 1137 days  in the early-intervention group and 1090 days  in the deferred-intervention group. Adjusting for follow-up period, participant age, ulcer size, and ulcer chronicity, there was no difference between the groups . Prespecified per-protocol analyses are presented in eFigures 4 and 5 in indicating that early intervention was, on average, the less costly strategy. Participants randomized to early intervention experienced, on average, greater QALYs after 3 years  using the EQ-5D-5L tariff recommended by National Institute for Health and Care Excellence. Early intervention was therefore a dominant strategy, with lower mean cost and greater mean QALY benefit. Findings were similar for 4-year and 5-year horizons (eTable 7 in Full details of resource use and costs for the 2 groups are presented in eTables 5 and 6 in 4 Longer-term follow-up in this study demonstrated that fewer recurrent ulcers per year of follow-up occurred in the early-intervention group, even though the time to first ulcer recurrence did not differ between the groups. The total mean costs were lower in the early-intervention group, and participants reported higher QALYs, indicating that early intervention is highly likely to be cost-effective irrespective of the willingness-to-pay threshold used by the health care system.One-year results from the EVRA trial showed that early ablation of superficial venous reflux accelerated healing of venous leg ulcers.16 The 4-year ulcer recurrence rates in this trial are comparable to those of previous studies evaluating ulcer recurrence after open varicose vein surgery, and outcomes in both groups of the EVRA trial are favorable compared with outcomes with compression alone.17 These findings support the strategy adopted in this study, where the choice of endovenous modality was left to the discretion of the treating clinician. Ablating superficial venous reflux is likely to be more important than the choice of modality.Ultrasound-guided foam sclerotherapy was the most common endovenous treatment in this trial, and some studies have reported high rates of technical failure compared with other endovenous modalities or open varicose vein surgery.The health economic benefits of early intervention demonstrated in this trial are particularly compelling because the premise of a less costly treatment strategy that offers more QALYs is an important driver for change in behavior irrespective of the country or health care system. The method of follow-up is a limitation of this study, as only telephone follow-up at a single time point after 1 year was possible owing to funding limitations; photographic assessment was not deemed feasible. However, most participants in this trial were kept under regular surveillance by recruiting centers as part of normal clinical care, resulting in accurately recorded outcome data. One-fifth of the participants in the deferred-intervention group did not undergo endovenous intervention at all. It is difficult to predict whether clinical outcomes would have been better if all participants had been treated, but delaying intervention was associated with fewer participants undergoing endovenous ablation. The results of this study reinforce the conclusions from the 1-year EVRA results, that early endovenous ablation of superficial venous reflux is highly beneficial for both patients and health care professionals. These observations indicate that a policy of deferred or delayed endovenous intervention is illogical for patients with venous ulceration.19 Although there may be a role for deep vein stenting in some patients with venous ulceration, excellent healing outcomes were achieved in the EVRA trial cohort with combined good compression therapy and superficial venous ablation. It should also be noted that patients with concomitant arterial disease, foot ulceration, or those not compliant with compression were not included.Long-term outcomes beyond 4 years remain unknown. Because chronic venous hypertension is multifactorial, ulcer recurrence is likely to be a common event in this population, despite endovenous ablation. Thirty percent of participants recruited to the EVRA trial suffered recurrent ulcers during follow-up. Aggressive investigation and treatment of venous outflow obstruction have been advocated, and the use of venous stents to correct nonthrombotic and post-thrombotic deep venous occlusive disease is increasing in popularity but requires robust evaluation.In this randomized clinical trial, early endovenous ablation of superficial venous reflux in addition to compression therapy reduced time to ulcer healing for primary ulcers. We found no statistical evidence that early endovenous ablation reduces time to first ulcer recurrence, but it was associated with a reduced incidence rate of recurrent ulcers and is highly likely to be cost-effective in the management of venous leg ulceration.


Caregiver burden also correlated significantly with PwP alexithymia (p < 0.001). However, caregiver burden was associated with sub-components “identifying feelings” (p < 0.008) and “external oriented thinking” (p < 0.004). These data support the importance of alexithymia as a non-motor symptom in PD.Parkinson’s disease (PD) is the second most frequent neurodegenerative disease of people who are beyond 50 years of age. People with PD (PwP) suffer from a large variety of motor and non-motor symptoms resulting in reduced health-related quality of life (HR-QoL). In the last two decades, alexithymia was identified as an additional non-motor symptom in PD. Alexithymia is defined as a cognitive affective disturbance resulting in difficulty to identify and distinguish feelings from bodily sensations of emotional arousal. In PD, the frequency of patients suffering of alexithymia is increased compared to healthy controls. The aim of the present study was to determine the relationship of alexithymia to HR-QoL of the PwP and caregiver burden of the corresponding caregiver. This cross-sectional questionnaire-based study used disease specific questionnaires for HR-QoL and caregiver burden. In total 119 PwP and their corresponding caregivers were included in the study. HR-QoL of the PwP correlated significantly with alexithymia ( Parkinson’s disease (PD) is a progressive neurodegenerative disease mainly affecting people beyond 50 years of age. In Germany, the prevalence of PD is estimated to be 0.5% of the population . People With regard to these symptoms, PD patients display deficits in recognition and definition of emotions, also called alexithymia . Taylor These characteristics are widely distributed, including a limited recognition of emotional faces and voices ,19,20,21The aim of the present study is use disease-specific questionnaires to investigate the association of alexithymia in PwP with health-related quality of life (HR-QoL) and the burden on the corresponding caregivers.This study was approved by the local Ethics Committee of Hannover Medical School . All patients and their caregivers gave their written informed consent to participate in the study. PwP and their caregivers were invited via the German PD association and the Hilde–Ulrichs Foundation. Local patients from the neurological wards and outpatient movement disorder clinic were included in this cross-sectional questionnaire-based study. Our sample included 119 PwP and related primary caregivers. Data from this patient and caregiver cohort unrelated to the current research question have already been reported elsewhere ,28.Inclusion criteria for PD patients were defined as neurologically determined Parkinson’s disease according to the MDS diagnostic criteria for PD with a duration of at least one year after onset of motor symptoms . PatientCaregivers and PwP were first asked to provide general background information .PwP then completed the Movement Disorders Society unified Parkinson’s disease rating scale (MDS-UPDRS) part II to evaluate their impairment of daily living ,31. MDS-To assess health-related quality of life (HR-QoL), PwP were requested to answer the Parkinson’s disease questionnaire 8 (PDQ-8) . PDQ-8 iCaregiver and patient depressive mood was assessed by Beck’s depression inventory (BDI) . CaregivAlexithymia of PwP and caregivers was measured by the Toronto Alexithymia Scale with 26 items (TAS-26). Participants responded to each item on a five-point Likert scale. Sum scores were divided by the number of completed items to obtain average scores for the total scale and three facets/subscales assessing different domains of alexithymia . Participants with a converted total score of >3 were considered alexithymic according to the manual ,37.n . To test whether any predictors explained unique variances in our outcomes, we included significant predictors in a multiple regression model for patients’ HR-QoL and caregiver burden. Further, we first included TAS-26 total score in the multiple regression analysis when the total score showed a significant correlation, we replaced the total score by significant facets  from the linear regression analysis. For the statistical analyses, SPSS 25.0  was used.Linear regression analyses were used to examine potential predictors of patients’ HR-QoL (PDQ-8) and caregiver burden (PDCB). To correct for multiple tests, the level of significance was set to α = 0.05/First, linear regression analyses were performed to test whether variance of health-related quality of life (PDQ-8) of PwP could be explained by alexithymia of the patient measured by TAS-26. Disease duration, MDS-UPDRS II, BDI, TAS-26 total, TAS-26 subdomain F1 “identifying feelings” and subdomain F2 “difficulties describing feelings” correlated significantly with PwP quality of life (after correction for multiple comparisons). Interestingly, age and TAS-26 subdomain F3 “external oriented thinking” did not show a significant correlation with PwP quality of life. All correlations are presented in p < 0.05). To correct for confounder and co-correlations, all significant patient variables were included in a multiple regression analysis. In this analysis, only MDS-UPDRS II reached statistical significance  displaying more depressive mood and quality of life restrictions. Additional, alexithymia correlated significantly with PwP HR-QoL, which was also found in other studies .In patients with dystonia, a significant correlation of alexithymia and HR-QoL was recently shown by our group . There aMany brain regions associated with alexithymia receive dopaminergic innervation as the frontal cortex and limbic structures . The priIn our study, we report a weak association of PwP alexithymia as psychological personality trait and caregiver burden. Until recently, there has been only reports in a small group of PD patients . In our Until recently, no intervention in PwP has been described to decrease alexithymia. Dafsari et al. reported minor effects of subthalamic nucleus deep brain stimulation reducing alexithymia of PwP . HoweverFor the recruitment of this large group of PwP and their caregivers, we decided to use a cross-sectional questionnaire-based approach. By this evaluation, we were only able to show associations and could not confirm causality. Further, because we used a questionnaire-based approach, we did not have access to extensive clinical characterizations of PwP, including the complete MDS-UPDRS ,56. ThesThe association of cognitive deficits and alexithymia would be interesting and will be a component of future clinical trials of our group. It seems that PD patients with alexithymia might not show cognitive deficits in general compared to non-alexithymic patients . HoweverThis study was not designed to analyze the impact of different dopaminergic treatment on alexithymia . In futuThis study provides evidence that alexithymia of PwP is associated with decreased HR-QoL and increased caregiver burden. Since the proportion of alexithymia is markedly increased in PwP, it may be considered as an additional non-motor symptom in PD. This study suggests more robust evaluation for assessing the impact of alexithymia adjusting for possible confounders. Interventions to decrease alexithymia in PwP are needed. These treatments might include psychotherapy and mindfulness-based interventions.