This latter group had multiple  episodes, and two patients had >1000 episodes. There were no clinical, echocardiographic, or laboratory findings able to discriminate patients with SAF from those in sinus rhythm in univariate and multivariable analyses; of note is that the Class C patients showed a higher diastolic blood pressure, resting heart rate, and indexed LA volume. Conclusions. Over a median of 13 days of Holter monitoring, the SAFARI study confirmed the usefulness of small wearable devices in detecting SAF episodes in real-world outpatients at risk for, but with no prior history of, AF.Background: The detection of subclinical/silent atrial fibrillation (SAF) in the general population is of the utmost importance, given its potential adverse consequences. Incident AF has been observed in 30% to 70% of patients with implanted devices, but its prevalence may indeed be lower in the general population. The prospective, multicentric, observational Silent Atrial Fibrillation ANCE Research Initiative (SAFARI) study aimed at assessing the SAF prevalence in a real-world outpatient setting by the means of a small, wearable, prolonged ECG Holter monitoring (>5 days) device . Methods: Patients ≥ 55 years of age at risk for AF were screened according to the inclusion criteria to undergo prolonged 3-lead ECG Holter monitoring. SAF episodes were classified as follows: Class A, <30 s; Class B, 30 to 299 s; and Class C, ≥300 s. Results: In total, 119 patients were enrolled  years). At a median of 13.5 (IQR 5–21) days of monitoring, SAF episodes were found in 19 patients (16%). A total of 10,552 arrhythmic episodes were registered, 6901 in Class A (n = 7 patients), 2927 in Class B (n = 3), and 724 in Class C (n = 9), (Class A vs. B and C,  Atrial fibrillation (AF) is associated with an increased risk of stroke and vascular dementia, especially in the elderly. The identification of AF in the general population is paramount, given its potential sequelae in terms of death and stroke occurrence ,2,3,4.Early studies have demonstrated that paroxysmal AF can occur even in the absence of symptoms and/or specific predictors, because of the unpredictability of situational/emotional stressors, physical activity, and other triggers, especially in subjects over 60 years of age ,3,4,5. MIncident AF has been estimated to occur in 30 to 70% of patients with permanent device monitoring, who are presumably affected by advanced heart diseases. However, its prevalence might be lower in the general population ,9,10,11.Current guidelines reinforce the need for detecting SAF, especially in those settings at a higher risk of cryptogenic stroke and in patients aged >55 years ,8. VarioThe Silent Atrial Fibrillation ANCE-Sicily Research Initiative (SAFARI) study aimed at investigating the SAF prevalence in a real-world, clinically stable, cardiovascular patient population, by the means of prolonged external ECG Holter monitoring (>5 days).Cardiologia Italiana del Territorio) Society (https://www.ancecardio.it (accessed on 3 August 2023).The SAFARI was a prospective, multicentric, observational study enrolling outpatients referred for stable cardiovascular disease, either as a de novo visit or follow-up. This study was conceptualized by the Sicilian Section of the Nationwide Scientific Society ANCE ;Class B: SAF episodes lasting from 30 to 299 s;Class C: SAF episodes lasting ≥300 s.SAF episodes were defined as the occurrence of irregular R-R intervals in the absence of detectable P-waves and classified as follows:Each researcher was given 2 devices, model CGM HI 3 LEADS ECG devices . Each one was associated with a smartphone  equipped with the CGM CARE MAP mobile app. Through a Bluetooth connection, the ECG signal was unceasingly conveyed to the smartphone and then to the WEB platform CGM CARE MAP, medical device class IIA . ECG files were available for online visualization and analysis by each researcher and the Central Core Lab  using deBefore starting the patient enrolment, each researcher attended a 4 h training session by two expert technicians of the CGM TELEMEDICINE industry. The live session was aimed at optimizing the management of the device, mobile phone, facilities, and tools, as well as the online software platform to register the patient data, analyze and elaborate the ECG file records.Echocardiography was performed using different machines equipped with 1.5–3.75 MHz transducers, with a thorough assessment of conventional measurements, chamber size, systolic and diastolic function, and valvular flow, according to current recommendations .Resting or exertional palpitations, dyspnea, and/or chest pain were assessed during a medical history interview, along with exercise tolerance, daily life limitations, and New York Heart Association (NYHA) class.This study was conducted in accordance with the principles stated in the Declaration of Helsinki. Written informed consent was obtained from all the participants at the time of performing the device testing. Ethical approval was granted on 15 February 2022 by the Institutional Review Board of the Paolo Giaccone University Hospital of Palermo, Palermo  (#02/2022). Members of the Steering Committee and the full list of the ANCE SAFARI Study group investigators are reported in p < 0.05. The analyses were performed using the IBM SPSS Statistics software, version 25.The data are reported as median and inter-quartile range (IQR) or mean ± standard deviation as appropriate for continuous variables, and number and percentage for categorical variables. The Mann–Whitney U test, ANOVA analysis of variance, Bonferroni or chi-square, and Fisher’s exact test were used. We also applied linear regression and binary logistic Wald stepwise backward regression to examine the association between the clinical, laboratory, or echocardiographic findings and the occurrence of AF. The null hypothesis was rejected at two tails for A total of 145 patients were screened. In total, 10 patients were excluded because of severe mitral or aortic valve disease (n = 3), severely depressed left ventricular (LV) function (LVEF < 35%) (n = 4), and permanent AF or a previous history of paroxysmal AF (n = 3). After enrolment, 16 patients from the remaining 135 were excluded due to missing laboratory data or ECG monitoring lasting <5 days, due to patch intolerance in spring and summertime. The final study population consisted of 119 patients (64 men), with a median age of 71 (IQR 55–85) years, whose demographic and clinical characteristics are reported in The median length of the ECG Holter monitoring in the study population was 13.5 (IQR 5–21) days. A total of 10,552 arrhythmic episodes were recorded. As expected, Class A episodes (n = 6901) occurred more frequently than Class B (n = 2927) and C (n = 724) episodes . Nine ouThere were no clinical, echocardiographic, or laboratory findings able to discriminate patients with SAF from those in sinus rhythm (SR) in the univariate and multivariable stepwise backward regression analyses. Of note is that a history of atypical chest pain, palpitations, and dyspnea were similar between the groups. Beta blocker use was more frequent in the SAF group .The main clinical and echocardiographic characteristics of the SAF group are displayed in The CGM HI 3-Lead ECG device is a small and easy-to-wear tool, with good-quality three-channel ECG recordings. As for similar devices, interferences and instability of the ECG trace occurred in the case of sweat and moisture on the electrodes or in the case of incidental patch detachment. In late spring and summertime, most patients (mainly women) complained of patch-related skin irritation and redness after three days of monitoring.This problem needed to be addressed upon electrode patch removal or new device application, so the decision was made by the Steering Committee to pause the study from 30 June to 10 September 2022. Enrolment was restarted thereafter. An important weakness of the device was a cracking tendency of the central rubber element devoted to covering the electrical connections, mainly occurring upon removal by the patient, such as when taking a shower or at the end of the monitoring. This problem often implied an interruption of the ECG trace recording and needed a replacement of the device.The SAFARI study investigated the incidence of SAF in a real-world setting of outpatients with chronic cardiovascular disease. A total of 10,552 atrial arrhythmic episodes were recorded in 19/119 patients (16%), distributed across three classes according to the length of the episodes. No clinical, laboratory, or echocardiographic predictors were identified in the multivariable analyses. Sustained SAF episodes (Class C) were observed in nine patients (7.6%); notably, these subjects had a larger indexed LA volume, as well as a higher heart rate at baseline, as compared to those from Classes A and B.Of note is that a not trivial proportion of the SAF patients complained of palpitations and atypical chest pain, so that the “subclinical” definition may not strictly be accurate.Although previous studies on incident AF have been performed with similar ECG-monitoring devices, the characteristics of patients, study design, protocols, and use of device, as well as the study timeline, were quite different, and this may hinder the generalizability of the results ,14,15,20In 2019, Wasserlauf et al. examined 31,349 h of smart-watch recordings in 24 ambulatory patients and showed a high sensitivity for AF detection and assessment of AF duration, with a positive predictive value of 39.9% and optimal patient tolerance . PatientOur results are consistent with a recent study testing the myBeat device on 150 patients, in which, at a median time of 4.95 ± 0.03 days, newly detected AF episodes occurred in 7.7% of the patients, and the rate of AF detection was significantly higher in the myBeat group (10.7%) than in the controls (4.7%) .p < 0.001) and sudden death . As the patients with AF were divided into subgroups with paroxysmal AF or non-paroxysmal AF, only paroxysmal AF was identified as an independent prognostic factor, irrespective of whether the AF was subclinical or not [As expected, in arrhythmic disease settings, the occurrence of SAF and related thromboembolic risk is greater than that in the general population. In 430 patients with hypertrophic cardiomyopathy (HCM), Yashiro et al. showed that AF was an independent determinant of HCM-related death , and the authors pointed out the fact that 2 weeks of ECG monitoring was the most useful timeline for the detection of AF recurrences in outpatient populations [The chance detection of paroxysmal AF increases with the length of monitoring system. Recently, 115 post-ablation patients (median age of 64 years) were investigated using AliveCor Kardia ECG recording and Holter monitoring. The two systems detected AF recurrences in 25.2% and 14.8% of the patients, respectively  have been reported to be associated with thromboembolism ,29, episSeveral limitations of this study should be acknowledged. The relatively small sample size limits the generalizability of the present results. As brain MRIs or CT scans were not available for all the patients, we were unable to perform a stroke risk analysis. Echocardiographic findings were available for 85% of the patients.Although overall reliable alerts were given when arrhythmic events occurred, false positive and negative episodes might occur in patients with low-voltage p waves or muscle interferences. Studies with larger cohorts and longitudinal follow-ups are ongoing and expected to shed further light on this topic .Over a median of 13 days of ECG Holter monitoring, SAF episodes were detected in 16% of our study population, with a duration of >300 s in 7.6% of cases. The SAFARI study confirmed the usefulness of small wearable devices in detecting subclinical arrhythmias in real-world outpatients at risk for, but with no prior history of AF. The prognostic impact of SAF episodes still needs further clarification.


Patients with high myopia have an increased lifetime risk of complications. The prevalence patterns of high myopia in children and adolescents in southern China are unclear. Early identification of high-risk individuals is critical for reducing the occurrence and development of high myopia and avoiding the resulting complications.This study aimed to determine the prevalence of high myopia in children and adolescents in southern China via real-world screening data and to predict its onset by studying the risk factors for high myopia based on machine learning.This retrospective school-based study was conducted in 13 cities with different gross domestic products in southern China. Through data acquisition and filtering, we analyzed the prevalence of high myopia and its association with age, school stage, gross domestic product, and risk factors. A random forest algorithm was used to predict high myopia among schoolchildren and then assessed in an independent hold-out group.There were 1,285,609 participants , of whom 658,516 (51.2%) were male. The overall prevalence of high myopia was 4.48% (2019), 4.88% (2020), and 3.17% (2021), with an increasing trend from the age of 11 to 17 years. The rates of high myopia increased from elementary schools to high schools but decreased at all school stages from 2019 to 2021. The coastal and southern cities had a higher proportion of high myopia, with an overall prevalence between 2.60% and 5.83%. Age, uncorrected distance visual acuity, and spherical equivalents were predictive factors for high myopia onset in schoolchildren. The random forest algorithm achieved a high accuracy of 0.948. The area under the receiver operator characteristic curve (AUC) was 0.975. Both indicated sufficient model efficacy. The performance of the model was validated in an external test with high accuracy (0.971) and a high AUC (0.957).High myopia had a high incidence in Guangdong Province. Its onset in children and adolescents was well predicted with the random forest algorithm. Efficient use of real-world data can contribute to the prevention and early diagnosis of high myopia. Patients with high myopia have an increased lifetime risk of complications such as cataract formation, retinal detachment from retinal holes or tears, myopic foveoschisis, peripapillary deformation, dome-shaped macula, myopic choroidal neovascularization, and glaucoma . The numIn the clinic, parents usually ask questions such as “will my child develop high myopia in the coming year” and “what is the possibility of the myopia of my child developing into high myopia in the future” even though their child has undergone only one ophthalmic examination. While it is well known that the risk of high myopia is relatively high in children with myopia onset during early school age , predictThe answer could be obtained by evaluating real-world screening of large-scale data. China promulgated guidelines for myopia prevention and control in children and adolescents in 2021 , emphasiArtificial intelligence (AI) may help in obtaining useful information from this screening data. Wang et al  trained Our study describes the prevalence patterns of high myopia from vision-screening data obtained from millions of children and adolescents in southern China. It also illustrates the method for using real-world screening data in research. An AI model aided in identifying the risk factors for high myopia occurrence and predicting the onset of high myopia at an early stage with high accuracy using real-world data for the first time.This retrospective and school-based study was approved by the institutional review board or ethics committee of Sun Yat-sen University, Guangzhou, China (2021KYPJ185), and was registered with the Chinese Clinical Trial Registry . The need for consent was waived because all data sets were deidentified before transfer for data analysis.Myopia screening was conducted in Guangdong Province, southern China, which included 21 cities with 115.21 million permanent residents. Guangdong’s gross domestic product (GDP) increased to 12,436.967 billion in 2021 . Using eThe staff of the participating schools received training from the administration, and local experienced certified optometrists performed the ocular examinations. UDVA was assessed monocularly using a Chinese standard logarithm VA chart (GB11533-2011) in an illuminated cabinet . LogMAR VA scores were recorded. Eyesight was measured at least thrice without cycloplegia using Topcon KR-1 . Measurements were repeated if one measurement deviated from the other two by >0.5 D. Three reliable measurements were averaged for analysis.Our screening data encompassed 13 cities in Guangdong Province . The cities have different populations, GDPs, and locations that show broad statistical variation as representing the whole province. Children and adolescents aged 6-20 years were recruited between 2019 and 2021. The right eye was arbitrarily selected to represent a specific individual. Individuals with missing records, other eye diseases, or eye surgeries were excluded.Noncycloplegic SE was calculated in accordance with the standard formula: SE = sphere + ½ × cylinder. Screening myopia was defined as a UDVA of ≥0 logMAR and an SE of ≤–0.5 D. It was further subdivided into low myopia (–6 D<SE≤–0.5 D) and high myopia (SE≤–6 D) according to the IMI myopia definition and classification . In the To visualize the relationship between high myopia and per capita GDP in cities in Guangdong Province from 2019 to 2021, we applied the random oversampling method to balance the distribution in data set 2. By selecting the ratio of primary school to middle school to high school as 2:1:1 in each city, we successfully obtained a newly balanced data set 3.Additionally, 2657 participants with high myopia and complete data were selected from data set 2 to determine risk factors and form data set 4. The SE value of the right eye was used in multivariable linear regression analysis. Six factors, namely age, sex, BMI, MAP, sexual maturity, and per capita GDP, included in the screening data, were selected as potential risk factors. Permutation importance was used to measure the influence of the factors on the predicted results.In data set 2, a total of 16,760 participants with a 3-year examination period were selected, of whom 380 participants presented high myopia during observation and 14,948 did not present high myopia in the 3 years. Considering this imbalance between participants (approximate 1:39), we randomly selected 1000 records from 380 subjects with replacement (random oversampling) and 1000 records from 14,948 subjects without replacement (random undersampling) to reach a new balance. The main outcome was high myopia onset, scored as 0 or 1, with 0 representing negative samples and 1 representing positive samples. Finally, data set 5 was formed, which included 2000 records to predict the onset of high myopia.Data set 5 was randomly split into a training set and a test set at a 4:1 ratio; a 5-fold cross-validation (CV) approach was used during training. Several machine learning algorithms, including logistic regression, a support vector machine, a k-nearest neighbor algorithm, and a random forest algorithm were applied to derive the classification results. Accuracy, precision, and recall were calculated to assess the prediction results for the training, CV, and test sets, respectively. Precision represents the percentage of true positive predictions among all positive samples, whereas recall indicates the percentage of true positive predictions among all correct predictions. Since we aimed to raise an early warning for cohorts at risk, recall was more persuasive than precision. The receiver operator characteristic (ROC) curve described the relationship between the true and false positive rates by setting different probability values as classification thresholds. A larger area under the ROC curve (AUC) indicated better efficacy. The Kolmogorov-Smirnov (K-S) curve assessed the model’s performance. The largest distance between the 2 curves  was calculated for the train, CV, and test sets separately. Finally, the model’s performance was validated by applying it to an independent hold-out group from Shenzhen Eye Hospital. These records, available for external validation, were obtained from 2547 individuals with a 3-year follow-up from 2019 to 2021 , of whom 47 subjects exhibited high myopia and 2500 did not during the 3 years of observation.t tests, 1-way ANOVA, the Kruskal-Wallis test, or the chi-square test, as appropriate. All P values were 2-tailed, where P<.05 was statistically significant.Data were processed and analyzed using the R programming language  and PythP<.001), but not in 2020 (P=.70). The overall prevalence of high myopia was 4.48% (2019), 4.88% (2020), and 3.17% (2021).In total, 1,285,609 participants were obtained in the 3 years of screening . There wP<.001). However, the rate of high myopia declined annually. Specifically, the prevalence over this period decreased from 1.33% to 1.08%, 5.88% to 5.02%, and 12.22% to 11.78% among participants in primary, middle, and high schools, respectively ; in first-level cities, it declined from 4.28% in 2019 to 1.12% in 2021 (P<.001). However, high myopia in third-level cities increased from 2.46% (2019) to 3.33%  but was stable in second-level cities between 5.03% and 5.38%.There were significant differences in the prevalence of high myopia in cities with different per-capita GDP levels. The high myopia rate in cities with the second-GDP-level cities was significantly higher than that in the first- and third-level cities for all 3 years , with Guangzhou  having a rate of 3.93% .of 3.93% . HoweverBecause MAP, GDP, sexual maturity, BMI, and sex reduced the model’s predictive performance, age, UDVA, and SE of the first 2 years were selected as predictors. T-distributed stochastic neighbor embedding showed that the data points were separable, thus revealing the feasibility for prediction . CompareReal-world research is important for public health and epidemiologic investigations . Our stuNCR was the main outcome of our research, which corresponds to Chinese large-scale myopia screening practices. Next, we included the combination of UDVA and NCR as a cutoff to screen for high myopia in large-scale school screening, aiming to reduce the gap between screening myopia (based on NCR) and its real-world prevalence. We acknowledge that the gold standard for refraction in children and adolescents is refraction with cycloplegia . TherefoAs confirmed in our study, the prevalence of high myopia in children and adolescents from southern China was higher than the global prevalence (4.0%) . The incWe also found a relationship between high myopia and economic development. A published report has shown that economic growth is negatively associated with the vision of school children in China. Every 100% increase in GDP was associated with a 20% increase in the relative risk of moderate to severe visual impairment . GuangzhWe extracted factors such as age, sex, location, MAP, BMI, and sexual maturity from the data set for further analysis and found that age was the most important risk factor, followed by MAP, GDP, sexual maturity, BMI, and sex. Behavioral and environmental factors such as economic development, time spent outdoors, physical activity, green scenery exposure, sunlight exposure, near-work activity, smartphone overuse, diet, and sleep, which have been reported as the main factors of myopia onset and progression ,22-24 caTo make full use of large-scale data from real-world vision screening and to provide immediate and timely warnings for high myopia onset, we studied the influence of related factors on high myopia incidence based on machine learning. Age was the most significant risk factor. MAP, GDP, sexual maturity, BMI, and sex affected high myopia, but they were not significant predictors and they reduced the predictive performance of the model. Finally, 3 variables—UDVA, SE, and age—were included to develop a random forest algorithm to predict the onset of high myopia in the following year. The results showed that prediction accuracy was >95%, which was reasonable and clinically acceptable. Although Lin’s , showingThe strengths of our study include the large sample size and the real-world nature of the data. Owing to some weaknesses of internal validity, study design, incomplete or incorrect data entry, or selection bias , many chHigh myopia had a high incidence in Guangdong Province. Its onset in children and adolescents was well predicted by the random forest algorithm. Efficient use of real-world data can contribute to the prevention and early diagnosis of high myopia. In the future, we aim to develop a smartphone app that allows people to input their age, UDVA, and NCR to predict the risk of high myopia. The adaptation of network- and smartphone-based myopia prediction platforms will provide a new model for vision screening, and it can dramatically improve vision health in the subsequent generations.


Motorist’s vestibular disorientation syndrome (MVDS) is a disorder in which patients experience dizziness while driving. MVDS is under-reported in the literature, and in clinical practice, it often goes unrecognized. We identified clinical characteristics of patients with MVDS using data from 24 patients who faced difficulties while driving and were diagnosed with MVDS. Their symptoms, duration of illness, precipitating factors, co-morbidities, history of other neuro-otological disorders, severity of symptoms, and associated anxiety and depression were reviewed. Ocular motor movements were recorded using video-nystagmography. Patients with vestibular disorders that can cause similar symptoms while driving were excluded. The mean age of the patients was 45.7 ± 8.7 years, and most were professional drivers (90.5%). The duration of the illness ranged from eight days to ten years. Most patients presented with disorientation (79.2%) exclusively while driving. The most common triggers for symptoms were higher speeds, i.e., >80 km/h (66.7%), multi-lane roads (58.3%), bends and turns (50%), and looking at other vehicles or signals while driving (41.7%). A history of migraines was reported in 62.5% of the patients, and motion sickness was reported in 50% of the patients. Anxiety was reported in 34.3% of patients, and 15.7% had depression. The video-nystagmography did not show any specific abnormalities. Patients responded to drugs used in prophylactic treatments for migraines such as Amitriptyline, Venlafaxine, Bisoprolol, and Magnesium, and to Pregabalin and Gabapentin. Based on these findings, a classification system and a diagnostic criterion for MVDS were proposed. Driving a vehicle in the dynamic environment of modern highways is a learned skill that reflects spatial cognitive ability ,2 and inThe vestibular labyrinth provides information about linear and angular acceleration of the head to central areas of the brain such as the brainstem, cerebellum, and vestibular cortex. It detects and measures movements in response to self-motion (active motion) and externally induced forces (passive motion) and is a crucial component in regulating gaze stability and postural reflexes . The vesThe vestibular system plays a vital role in spatial navigation and head orientation by judging the speed of head movement. The visual system provides input regarding the differential movement of the visual field (optic flow) during driving, which is important due to the paucity of visual cues during driving . AsymmetThe vestibular, visual, and proprioceptive systems coordinate with each other to adjust outgoing motor responses contributing to balance, posture, and stabilization of gaze. An impairment in balance can arise due to central or peripheral components of the vestibular system that project an illusion of self-motion like dizziness or vertigo and disorientation in space .Vestibular symptoms as a passenger, i.e., motion sickness, are a well-known entity. Vestibular symptoms can also happen while driving in some vestibular disorders such as vestibular migraines, Meniere’s disease, etc. . In 1985The term motorist’s vestibular disorientation syndrome (MVDS) was coined to describe these symptoms ,7. SimilIn clinical practice, MVDS, though less common, is not unfamiliar. However, since its first description in 1985, literature exploring the syndrome’s characteristics has seldom been reported, making it an understudied and underreported phenomenon. As there is a rising concern about the possibility of accidents endangering the safety of patients with MVDS while driving, there is a need to understand the patient characteristics of MVDS to aid in early diagnosis and treatment.This study provides insights into clinical features and video-nystagmography (VNG) findings in MVDS patients. VNG findings help in making a clinical diagnosis by recording eye movements in graphical as well as video recording formats, and aid in differentiating between a central or peripheral vestibular lesion ,12. We aA total of 24 patients presenting with signs and symptoms of MVDS, who approached the vertigo clinic in Dubai, United Arab Emirates, and Cochin, India, between January 2018 and September 2021 were enrolled in the study.DisorientationFalse perception of the vehicle turning to one sideSweating, Palpitations, cold extremitiesStiffness of bodyPatients presenting with one or more symptoms of dizziness while driving, including:Higher speed (>80 km/h)Bends and turnsMulti-lanesUpward or downward slopesLooking down from bridgesOvertakingLooking at other vehicles while drivingClosed tunnelsSymptoms present exclusively while driving and aggravated during the following situations:Symptoms leading to significant distress or functional impairment while drivingSymptoms that were not better attributed to another disease or disorderPatients presenting with the symptoms listed below were included in the study:Patients having symptoms of visual vertigo de novo or due to vestibular migraine or PPPD without driving were excluded from the study. Patients manifesting other vestibular diseases such as Meniere’s disease, BPPV, etc., and those who experienced dizziness exclusively as a passenger (motion sickness) were excluded from the study.The medical records of 24 patients diagnosed with MVDS were reviewed in detail. Their demographic data, including age, sex, occupation, symptoms, duration of symptoms, precipitating factors, co-morbidities, history of neuro-otological disorders, and psychiatric illnesses were recorded.The symptom severity was assessed using a 25-item self-reported Dizziness Handicap Inventory (DHI) . PatientOcular motor movements were recorded using video-nystagmography goggles . The tracing obtained were of horizontal and vertical eye movements with simultaneous recording for eye video. Brain MRIs and other vestibular tests were performed as needed.Patients who met the inclusion criteria and were diagnosed with MVDS and treated accordingly. A variety of drugs based on co-morbidity were used, such as Amitriptyline, Bisoprolol, Magnesium, Venlafaxine, Desvenlafaxine, Gabapentin, and Pregabalin. The response to each treatment modality was recorded during follow-up visits. Patients underwent assessments after one month of the treatment and during subsequent follow-ups using the Dizziness Handicap Inventory. The natural history of the symptoms was also recorded.Data were analyzed using SPSS Statistics for Windows, Version 20.0 , and Microsoft Excel 2016. Categorical data including demographics, clinical characteristics, and video-oculography findings were summarized as frequency and percentage. In contrast, continuous variables such as smooth pursuit and optokinetic asymmetry values were summarized as mean, median, and quartiles.A total of thirty patients who reported dizziness while driving were screened for MVDS. However, three patients were diagnosed with vestibular migraine, two with PPPD, and one with Meniere’s disease. These patients also had symptoms without driving and were therefore excluded from the study. Among the remaining 24 MVDS patients enrolled in the study, 21 were recruited from the UAE and three from India. The mean age of the patients was 45.7 ± 8.7 years. Most of the patients were professional drivers (90.5%) with a minimum driving experience of 5 years.Among the patients, 70.8% reported experiencing symptoms until they pulled over, and in 45.8% of the patients, the symptoms lasted for 1 min to 20 min. Motion sickness as a passenger was reported in 50% of the patients. The severity of dizziness reported by patients on a scale of 1-10 had a mean score of 6.1 ± 2. When asked about the trigger of these symptoms, most patients reported higher speeds (>80 km/h) (66.7%), multi-lane roads (58.3%), bends and turns (50%), looking at other vehicles or signals while driving (41.7%), and other factors. Out of the 24 patients, nine (37.5%) felt that the vehicle veered to the right side, eight (33.3%) felt it veered to the left, and seven (29.2%) did not feel any veering. The majority (62.5%) of the patients did not report any diurnal variation in symptoms.The PPPD score based on the Niigata questionnaire ranged from 0 to 27, with a median  score of 2.5 . None of the patients had a high enough visual score (>9/24) to qualify for visual vertigo or PPPD. While most patients did not have anxiety (65.2%), 17.4% had severe anxiety when assessed using the GAD-7. Similarly, the majority did not show any depressive symptoms (83.3%), as evaluated by the PHQ-9.Video-nystagmography (VNG) or video-oculography recordings of 17 patients are summarized in Normal horizontal saccades were observed among all the patients, but 11.8% had hypometric vertical saccades. The horizontal and vertical smooth pursuit was normal in 76.5% and 5.9% of patients, respectively. Up-beating spontaneous nystagmus was observed in three patients (17.6%), and three (17.6%) had nystagmus induced by head shaking and hyperventilation.During gaze testing with fixation, none of the patients had any abnormalities. During gaze testing without fixation, 20%, 13.3%, 12.5%, 12.5%, and 13.3% patients had nystagmus in the centre, left, right, up, and down gaze, respectively .We also recorded horizontal smooth pursuit (HSP) and optokinetic (OK) asymmetry of the patients. Those who felt the vehicle veering to the right had a higher mean (SD) HSP asymmetry of −4.6 (12.9) °/s and a horizontal OK asymmetry of −4.1 (6) °/s compared to those who felt the vehicle turning to the left.The mean difference (SD) in the DHI score before and after treatment was 35.5 (20.7) in four patients after treatment. Post-treatment DHI scores could not be recorded in the remaining patients who underwent treatment, as some of them were lost to follow-up and some did not respond to the questionnaire. However, we have recorded the subjective improvements among patients depending on the treatment provided .The present study was conducted to understand the clinical characteristics of patients presenting with motorist’s vestibular disorientation syndrome. Although the syndrome and its characteristics were identified in 1985 by Page and Gresty , it has To the best of our knowledge, this is the second study after Page and Gresty  to reporStopping or reducing the frequency of drivingAvoiding long journeysAvoiding multi-lane highwaysAvoiding driving on bridgesReducing overtakingReducing the speed well in advance of traffic signalsDriving at low speeds (less than 80 km/h) on single-lane roadsTaking frequent breaks on long journeysKeeping water/juice ready during the journeyMVDS patients in the present study were predominantly driving professionals who had the illness for a duration of 8 days to 10 years. Other studies have attributed this wide range of illness duration to a delay in diagnosis, which may be due to a lack of awareness of the disorder, motorists assuming it was an issue related to the car, and not knowing which doctor to consult ,9. PatieMVDS occurs as a result of inadequate or misinterpreted sensory signals related to orientation and motion in the space of the complex and dynamic driving environment  Most of Dizziness or disorientation was the most commonly reported symptom among patients in the present study . Visual Golding and Gresty et al. reported anxiety as a triggering factor for disorientation while driving . PsychiaPrevious case studies on MVDS patients ,7,17 didIn the present study, among the patients who were present on follow-up, the DHI score of 24 before treatment with Amitriptyline, Bisoprolol, and Magnesium, was reduced to 8 in one case. In another case, a score of 60 decreased gradually to 30, then 20, and finally to 2 when treated with Amitriptyline followed by Bisoprolol and Magnesium, and eventually replacing Amitriptyline with Desvenlafaxine. Amitriptyline, in another case, also reduced DHI scores from 52 to 4. One of the patients exhibiting vascular loops on an MRI brain scan was treated with Gabapentin, and their DHI score reduced from 36 to 16. However, we could not make a decisive conclusion due to the limited number of patients that were available on follow-up. We found that a few patients showed 80% improvement when treated with migraine medications such as Amitriptyline, Bisoprolol, Magnesium, or Desvenlafaxine. This link between MVDS and migraines needs to be explored in future studies .Considered initially as a subset of phobic postural vertigo, MVDS can be regarded as a task-specific functional neuro-vestibular disorder . Patient-Primary MVD, i.e., MVDS: symptoms are exclusively experienced while driving, and the patient has no other vestibular disorder to explain the symptoms.-○Secondary to visual vertigo in vestibular migraine○Secondary to visual vertigo in PPPD○Secondary to other vestibular disordersSecondary MVD: the patient has an underlying vestibular disorder explaining the symptoms, and the symptoms are not exclusive to driving.Patients can present with more than one vestibular disorder; hence, it is imperative to take a good history . In addiThe vehicle might turn to one sideDisorientationPre-syncopal symptoms Postural adjustments *Higher speed (>80 km/h)Bends and turnsMulti-lanesUpward or downward slopeLooking down from bridgesOvertakingLooking at other vehicles while drivingClosed tunnelsSymptoms are present exclusively while driving and aggravate during the following situations:The disorder usually begins insidiously without any identifiable precipitating event.Symptoms cause significant distress or functional impairment while driving.Symptoms are not better attributed to another disease or disorder **.We also propose the following diagnostic criteria for primary MVDS in patients presenting with two or more symptoms of dizziness while driving on most days, such as:* Such as stiffness of body, holding the steering wheel tightly** Patients devoid of de novo symptoms of visual vertigo or other vestibular disorders presenting while driving.There is limited literature on the treatment of MVDS. The general principles of MVDS treatment are the exclusion of organic disease, treatment of general state anxiety/phobia, explanation of symptoms, and progressive desensitization. One approach to treatment involves grouping patients into two categories :(1) Patients with car-tilt illusions and previous vestibular disorders without panic disorders should receive vestibular rehabilitation with progressive desensitization.(2) Patients with panic disorders without car-tilt illusions and vestibular disorders should receive selective serotonin reuptake inhibitors (SSRI), anxiolytics, and cognitive behavioral therapy (CBT).In our study, we treated patients with various medications such as Amitriptyline, Venlafaxine, Pregabalin, Bisoprolol, Gabapentin, Pregabalin, and Magnesium with variable relief of symptoms. The natural history of MVDS ranges from complete recovery with or without treatment to chronic suffering without relief.As this was a two-center study, there was variation in the approach to history taking between the centers. Therefore, data concerning symptoms, etc., were not recorded for a few patients. The treatments were not uniform across all patients, and the of follow-up outcomes after treatment were not recorded in all the patients. Because this was a retrospective study, gaps in the data could not be addressed by following up with patients; therefore, a prospective, cross-sectional study could help to accurately identify patient characteristics.MVDS is a distinct clinical entity that requires diagnostic consideration and treatment. The time to diagnose this condition can be reduced with widespread recognition. Most patients with MVDS have a history of migraines and non-specific video-oculographic abnormalities. We also propose a classification system, diagnostic criteria, and medical treatment options for MVDS.


Mast cell leukemia (MCL) is a rare and aggressive subtype of systemic mastocytosis (SM). MCL diagnosis requires fulfillment of diagnostic criteria for SM plus at least 20% immature/atypical mast cells on bone marrow smear. Based on different clinicopathological characteristics, MCL is subdivided into the following variants: primary (de novo) or secondary (from a prior SM form); acute with C-findings (where C-findings represent organ damage requiring cytoreductive treatment) or chronic without C-findings; and finally, MCL with or without an associated hematological neoplasm. The latter category was renamed MCL with an associated myeloid neoplasm in the 2022 International Consensus Classification (ICC). The relevance of the distinction between the leukemic and aleukemic forms, based on the percentage of circulating mast cells, is under discussion. In this study, we review the current knowledge on MCL with the aim of increasing clinicians’ and pathologists’ awareness of this rare, still incompletely understood disease with a grim prognosis. Mast cell leukemia (MCL) is the leukemic form of SM with at least 20% mostly immature mast cells on bone marrow aspirate. MCL may develop de novo, in the absence of a prior SM, or it may represent a progression from a previous SM. MCL may be sub-divided into the more frequent, aggressive acute form with signs of organ damage (C-findings) and the chronic form lacking C-findings and presenting a more stable course, although over time, progression to acute MCL is common. The 2022 WHO subtype of MCL with an associated hematological neoplasm was renamed MCL with an associated myeloid neoplasm in the 2022 International Consensus Classification (ICC). The relevance of the distinction between the leukemic and aleukemic forms based on the percentage of circulating mast cells is a matter of debate. The current knowledge on MCL is restricted mainly to single reports or case series with a limited number of larger studies. Our aim is to provide a comprehensive overview of this rare disease in terms of clinical manifestations, morphology, phenotype, molecular characteristics, differential diagnosis, outcome and treatment. A general overview on mastocytosis is also included. Mast cell leukemia (MCL), which was first described by Joachim in 1906, represents the most uncommon form of systemic mastocytosis (SM) and is seen in less than 5% of all SM patients ,8,9. It is characterized by the leukemic expansion of mostly immature mast cells (MCs) in the bone marrow (BM) and other organs, with organ damage and a poor prognosis in the majority of cases with a median overall survival (OS) of 1.6 years ,6,7,8,9.The diagnosis of MCL requires the presence of the diagnostic criteria for SM plus at least 20% atypical, immature MCs in BM smears. In the case of suboptimal BM smears, according to the 2022 International Consensus Classification (ICC), MCL diagnosis can be performed on BM biopsy, which is usually diffusely infiltrated by atypical/immature MCs  10]..10].Under discussion is the real importance of sub-dividing MCL into the leukemic variant with at least 10% atypical/immature MCs in the peripheral blood (PB) and the aleukemic variant with less than 10% circulating MCs .MCL is a rather heterogeneous disease that is sub-divided into an acute form and a chronic form on the basis of the presence or not of organ damage (C-findings). The acute form, occurring in the majority of patients (60–90%), has a rapid development and an aggressive course with massive organ damage; whereas the chronic form has a more prolonged course without rapid organ damage, although, over a variable length of time, progression to acute MCL seems to be the rule .In addition, the disease may develop de novo  or it may be secondary when an antecedent MC neoplasm is present; finally, MCL may occur alone or with an associated hematological neoplasm, the so-called MCL-AHN as per the 2022 WHO classification . This caDue to the rarity of MCL, both the pathogenesis and treatment of the disease are not well defined.Herein, we aim to highlight the clinicopathologic features of this aggressive and still incompletely known disease.Mastocytosis is a very heterogeneous group of neoplastic MC disorders, ranging from forms with an indolent clinical behavior such that patients may have an almost normal life-expectancy to other forms with an aggressive course and short survival due to multi-organ involvement ,15,16,17cutaneous mastocytosis (CM) with skin-limited disease, which is more common in children and which presents good biological behavior;SM with involvement of one or more extra-cutaneous organs, which is more frequent in adults and has a less favorable outcome;mast cell sarcoma (MCS), a very uncommon, localized and high-grade MC neoplasm ,20,21,22The 2022 WHO classification continues to recognize three main forms of mastocytosis :cutaneouAccording to the 2022 WHO classification, the diagnosis of SM requires that the major criterion plus one minor criterion or at least three minor criteria are fulfilled , whereasKIT mutation as minor diagnostic criteria ..19].However, tryptase and CD117 expression alone is not indicative of the neoplastic nature of MCs.The immunohistochemical expression of CD25 and/or CD2 is currently recognized as a minor diagnostic criterion of SM, as the expression of these markers is indicative of the clonal origin of MC infiltrates  [2].These two antigens are not expressed on normal MCs, myeloid precursors or immature MCs present in other myeloid neoplasms, including myelomastocytic leukemia (MML) .CD25 is considered a more sensitive marker compared to CD2, which is not found in all cases of SM and decreases with disease progression .In MCL, the phenotype of MCs is similar to that of SM, with MCs usually expressing tryptase, CD117, CD25, CD2, CD33, CD44 and CD9.However, there are some differences between ISM and MCL. Compared with ISM, the percentage of cases positive for tryptase and CD2 is lower in MCL, as the expression of both tryptase and CD2 decreases with malignant progression .The expression of CD2 is not detected in 48% of MCL, whereas CD25 expression is absent in 25% of MCL, and one third of MCL is reported to be negative for both CD25 and CD2.KIT D816V-positive MCL, the co-expression of CD25 and CD2 is more frequently found compared with KIT D816V-negative MCL (66% versus 25%).In CD52, HLA-DR, CD123 and CD30 are more often expressed in MCL compared with ISM.KIT-independent oncogenic events occurring when the disease progresses ..10].KIT mutations and/or other genetic abnormalities with SM, whereas KIT mutations are not found in lymphoid or plasma cell neoplasms co-occurring with SM [The associated myeloid neoplasms often share  with SM .Myeloid malignancies associated with SM may be myelodysplastic neoplasms (MDS), myelodysplastic/myeloproliferative neoplasms (MDS/MPN), AML and chronic myeloproliferative neoplasms (MPN).According to all the published data, chronic myelomonocytic leukemia (CMML), which is included in the group of MDS/MPN, is the most frequent myeloid neoplasm associated with SM ,23,36.All subtypes of AML may occur in association with SM, with a prevalence for AML types M2 and M5 .The association of SM with lymphoid or plasma cell neoplasms is rare (10% of cases) and, interestingly, monoclonal gammopathy of undetermined significance (MGUS) is more frequent in SM patients than overt plasma cell myeloma or lymphomas ,24.The diagnosis of SM-AHN (SM-AMN) represents a diagnostic challenge for pathologists, and it is based on a thorough evaluation of BM biopsies with the aid of appropriate immunostainings to identify MCs.In particular, a diffuse blast proliferation of AML may obscure a coexistent neoplastic MC infiltrate, which may be easily overlooked if appropriate immunohistochemistry is not performed. A second BM performed after induction chemotherapy for AML may reveal compact MC infiltrates which become evident due to the pronounced therapy-related aplasia of myeloid blast cells ,38. AML According to Horny et al., the incidence of SM-AHN (SM-AMN) would increase if appropriate immunostainings such as tryptase, CD117 and CD25 were applied routinely in all BM biopsies containing infiltrates of MDS, AML, CMML and MPN .It has been supposed that both SM and the associated hematological malignancy may originate from an early uncommitted hematopoietic cell with a subsequent progression into phenotypically different clones. This hypothesis is considered the most likely in cases of SM associated with myeloid malignancies, whereas the hypothesis of the coincidental occurrence of two different clonal hematological neoplasms may be more likely for the rare event of SM associated with lymphoid or plasma cell malignancies ,23,37,38The detection of SM associated with another hematological malignancy has important prognostic and therapeutic implications, as MC neoplastic proliferations are often resistant to the majority of cytoreductive drugs employed in myeloid neoplasms. Detecting both neoplasms is crucial to offer therapies that could target both diseases.In the 2022 MCL study by Kennedy et al., approximately 34% of patients had a diagnosis of MCL-AHN .The most common AHN was myelodysplastic syndrome/myeloproliferative neoplasm unclassifiable , followed by CMML (25.8%), chronic eosinophilic leukemia CEL; 9.7%) and AML (9.7%), whereas non-Hodgkin lymphoma and plasma cell myeloma were found in 3.2% of cases [.7% and AMML is a rare but probably understated type of leukemia characterized by an increase in immature MCs and metachromatic blasts in the context of an advanced myeloid neoplasm .If tryptase and CD117 were routinely applied in the diagnosis of all cases of advanced myeloid neoplasms, it is estimated that MML would be identified in 3% of myeloid neoplasms ,40.By definition, SM diagnostic criteria are not present in MML; in particular, the compact aggregates of SM are not identified in MML, CD25 is negative, and KIT mutations are absent.at least 10% immature MCs/metachromatic blasts in BM smear in the context of an advanced myeloid neoplasm;>5% myeloblasts in BM smear or PB (sign of advanced myeloid neoplasm);at least 10% of BM cells should be MCs (positivity for tryptase and CD117);absence of SM diagnostic criteria .The diagnostic criteria of MML are the following:KIT mutations are in favor of MCL diagnosis [The distinction between MML and MCL may be difficult; however, the presence of immature MCs expressing CD25 and the identification of iagnosis ,39,40. TMML needs to be differentiated also from tryptase-positive AML .This rare form of AML is characterized by an increased serum tryptase level similar to that of MCL and MML; however, in the majority of tryptase-positive AML, only tryptase and not CD117 is co-expressed by CD34-positive myeloblasts . On the MML needs to be separated from SM/AHN, in particular when the AHN is represented by AML. In patients with SM/AHN, the diagnostic criteria for SM need to be present, whereas by definition, they are absent in MML.KIT mutations are found in MML and acute basophilic leukemia. The diagnosis of acute basophilic leukemia needs to be further confirmed by the use of immunohistochemical stains for basophil-specific antigens such as BB1 and 2D7.Finally, both MCL and MML should be distinguished from acute basophilic leukemia and chronic basophilic leukemia ,39,40. AChronic basophilic leukemia is characterized by mature and often hypo-granulated basophils expressing tryptase, but not CD117. Blood basophilia is typically present in chronic basophilic leukemia and absent in MCL, acute basophilic leukemia and MML.The prognosis of MCL is significantly worse than other subtypes of SM, with an OS ranging from 0–5 to 2.6 years, and no therapy is capable of inducing a durable long-term response, although encouraging results have been obtained with midostaurin ,5,6,9.In a study by Kennedy et al., MCL patients had an OS that was significantly inferior (1.6 years) to patients with ASM (6.2 years) and SM-AHN (2.8 years) .The median OS of the entire large MCL cohort recently evaluated by Kennedy et al. was 1.6 years, although the following differences were present, depending on the MCL subtype .In detail, patients with MCL-AHN had an inferior OS compared to patients with MCL alone (1.3 versus 2.3 years); patients with leukemic MCL had an OS inferior to patients with the aleukemic form (0.4 versus 1.9 years); and patients with any circulating MCs had an OS inferior to patients with no circulating MCs (0.5 versus 3.2 years).No difference in survival outcomes was observed between patients with de novo MCL and patients with sMCL; by contrast, the presence versus absence of C-findings, which were used to define acute and chronic MCL, respectively, was highly significant, as acute MCL had an OS inferior to chronic MCL .KIT D816V-positive patients had superior OS compared to KIT D816V-negative patients (3.2 versus 0.9 years), and interestingly, patients with alternative KIT mutations had an OS similar to patients without KIT mutations.In addition, Kennedy et al. observed that S/A/R mutations reduces OS in MCL patients [As previously mentioned in the paragraph on MCL molecular features, it has been demonstrated that the presence of patients ,9,34.KIT D816V negativity, S/A/R mutations and treatment status (not receiving midostaurin) were all associated with inferior outcomes; whereas chronic MCL, KIT D816V positivity and midostaurin therapy at any point were associated with superior OS [In conclusion, the study by Kennedy et al., demonstrated that a diagnosis of MCL-AHN, the presence of any circulating MCs, abnormal karyotype, erior OS .S/A/R-positive cases observed in the German study compared with the American report [In the study of Jain et al. reporting a single-center American study, the OS was longer than in the German study by Jawhar et al. (31 versus 17 months) ,6. This n report ,6.For MCL, few therapeutic options are available, and due to the rarity of the disease, no approved standard therapy exists.Cytoreductive chemotherapy has been used for MCL treatment; most frequently, single-agent 2-CdA or multi-agent AML-type induction regimens have been used .A possible option for young and fit patients is represented by ASCT when a response to 2-CdA and/or chemotherapy is obtained . ASCT neHowever, because of advanced age, disease-related organ damage and poor response to chemotherapy, only a small proportion of patients is eligible for ASCT.In addition, only rarely complete remission is obtained after transplantation. In a retrospective report, it was observed that among advanced SM undergoing ASCT, MCL patients showed the worst outcome, with a three-year OS of 17% ,42.Encouraging results have been obtained with KIT-targeting drugs, particularly midostaurin.In 2017, midostaurin, a small-molecule inhibitor of multiple type III tyrosine kinase receptors, has been approved by the Food and Drug Administration (FDA) and by the European Medicines Agency (EMA) for patients with ASM, SM-AHD and MCL ,9,43.In the 2022 study by Kennedy et al., patients receiving midostaurin at any point during their treatment course had a superior median OS compared to patients non-treated with midostaurin (2.3 versus 1.1 years); the difference was even more significant for patients receiving midostaurin as a first line of treatment compared to patients who received a different therapy (3.2 versus 1.3 years) .KIT D816V inhibitor, was approved by the FDA as a first-line treatment in patients with advanced SM and in 2022 by the EMA as second-line therapy [In 2021, avapritinib, a selective  therapy ,45. The S/A/R mutations and KIT D816V negativity are associated with a worse outcome. Midostaurin treatment at any point in the course of the disease is associated with a better prognosis. Further studies are essential to improve clinicians’ and pathologists’ awareness of the disease in order to provide an accurate and rapid diagnosis with consequent appropriate treatment.MCL is a rare and aggressive form of SM. The current available data on the disease are based on single reports, with only four large studies published so far. Despite its overall poor prognosis, some characteristics are relevant to predicting MCL patient survival. In particular, a diagnosis of MCL-AHN, the presence of


Human immunodeficiency virus (HIV) infection is known to be associated with the development of Hodgkin’s lymphoma (HL). Exclusive extranodal bone marrow involvement is less common. Co-infection by other viruses, such as the Epstein–Barr virus (EBV), increases the incidence of a frequent complication denominated by hemophagocytic lymphohistocytosis (HLH). We present the case of a 50-year-old patient with the above clinical spectrum who develops several serious complications during treatment. Hodgkin’s lymphoma (HL) is a lymphoproliferative syndrome frequently associated with patients with human immunodeficiency virus (HIV) infection, although it is not currently recognized as a defining entity of human immunodeficiency syndrome (AIDS) . ExtranoA 50-year-old Uruguayan man with no personal history of interest presented to the emergency department with a four-month history of asthenia, anorexia, profuse nocturnal diaphoresis, weight loss of up to 10 kg, and intermittent fever with associated dystrophic sensation. The patient reported risky sexual relations, with the last serology screening for HIV infection three years earlier being negative. Relevant clinical findings on admission included a striking masseteric and temporal atrophy, oral thrush, bilateral laterocervical subcentimeteric lymphadenopathy, predominantly on the left side, residual lesions of metameric herpes zoster on the left side, and a palpable hepatosplenomegaly. In addition, the patient had a body temperature of 39 °C. 3/µL (0.9 × 103/µL lymphocytes and 1.9 × 103/µL neutrophils), hemoglobin (Hb) 10.8 g/dl, platelets (Pts) 134,000 × 103 µL, as well as an ultrasensitive C-reactive protein of 19 mg/dl. The hemogram showed pancytopenia (leukocytes (Leu) 3.16 × 10A full-body computed tomography (CT) scan showed a hepatomegaly of approximately 23 centimeters (cm), without focal lesions, as well as lymphadenopathies in the upper abdomen, hepatic hilum up to 29 millimeters (mm), and retroperitoneal and mesenteric up to 11 mm in subcentimeter iliac and inguinal regions.Flow cytometry was requested due to alterations in the hemogram showing severe lymphopenia at the expense of all natural killer, B, and T lymphocyte subpopulations, the last one presenting a significant decrease in the TCD4+ cell count (<100 cells/µL) with a preserved TCD8+ cell count. In addition, circulating lymphoplasmoid cells were identified without light chain restriction, suggesting normality. Based on these findings, blood and urine cultures were requested and were normal. A serology for multiple viruses showed positive HIV1 and HIV2 antibodies and p24 antigens. The confirmatory Western blot test showed positive antibodies against HIV1 gp160, gp41, and p24 proteins. Regarding viral load, quantification of HIV-1 viral RNA was 72,100 copies/mL (4.86 log) . Given the HIV infection, determination of lymphocyte populations was performed. The TCD4+ lymphocytes in absolute value resulted in 70 cells/microliter with a CD4/CD8 ratio of 0.14. Resistance mutation studies for HIV antivirals were also performed without finding any resistance to them.In addition, anti-core antibodies for hepatitis B virus were positive; the viral load was measured by DNA and the rest of the study was negative, presenting the patient as a carrier of this virus within the study. The rest of the study showed no relevant alterations with negativity for hepatitis C, syphilis, and tuberculosis. Leishmania serology was also performed due to the patient’s pancytopenia, with negative results.At that time, the patient started highly active antiretroviral therapy (HAART)  and empiric antibiotherapy, started at emergency department, was suspended due to the low clinical suspicion of bacterial infection. After HAART initiation, HIV viral copies decreased rapidly to 67 copies/mL (1.83 log) three months later and undetectable values after six months of treatment. CD4 lymphocytes reached levels above 100 cells/microliter eight months after HAART.Bone marrow (BM) aspirate was performed and showed no cytological data or dysplasia, infiltration by a lymphoproliferative syndrome, or plasma cell dyscrasia. Marrow blood immunophenotyping was normal, as were the marrow cultures. BM biopsy revealed isolated ample cytoplasmic cells with one or more nuclei with prominent nucleoli-expressing CD15, CD30, PAX5, and EBER as well as images of hemophagocytosis with hardly any residual normal hematopoietic tissue. Reed–Sternberg cells were observed. In addition, large numbers of polytypic plasma cells and a striking increase in CD3/CD8-positive small T lymphocytes and abundant loose epithelioid histiocytes were also shown, with no granuloma formation. These cells did not show expression for CD20 or CD79a. Furthermore, no human herpes virus 8 (HHV8) positive cells or an increase in the number of blasts were observed .These findings make the diagnosis of Hodgkin’s lymphoma (HL) more likely. In order to confirm and stage the disease, a positron emission tomography CT (PET-CT) scan was requested, revealing multiple supradiafragmatic and infradiafragmatic adenopathies, most of them with discrete–moderate fluorodeoxyglucose (FDG) uptake (some with intense activity). The one at the right inguinal region was the most accessible for biopsy .In addition, FDG uptake was revealed in the gastric fundus, showing only data of gastritis, hepatomegaly without significant alterations to the metabolism, and a discrete diffuse hypermetabolism in BM. No alterations were found in the rest of the study. A lymph node biopsy was performed showing hyperplasia of germinal centers with increased vascularization in the centers and interfollicular area with expansion and fibrosis of the capsule. Monocytic cells were revealed in subcapsular sinusoids, histiocyte clusters, and numerous plasma cells in the centers and perifollicular area that did not show light chain restriction. Abundant CD30 cells were revealed in the centers and interfollicular area with numerous EBV (EBER)-positive cells in the centers, although in smaller numbers, surrounding them. These findings were compatible with the histologic diagnosis of HIV-related lymphoid hyperplasia associated with possible EBV reactivation .As part of the etiological study, the viral load of Epstein–Barr virus measured by the DNA polymerase chain reaction (PCR) in peripheral blood using probes that hybridize in the EBNA-1 protein gene was requested and resulted in 47,406 copies/mL. With these findings, the striking clinical improvement after initiation of antiretroviral therapy and the rarity of Hodgkin’s disease isolated to marrow made us take the results of the first marrow biopsy with caution, delaying systemic treatment with chemotherapy.Later, four weeks after initiation of HAART, the patient’s clinical condition worsened, making HL the likely diagnosis. 3/µL, Hb 6.6 g/dL, Pts 42,000/µL), recurrence of fever, persistent splenomegaly on physical examination, hyperferritinemia , and hypertriglycidemia (275 mg/dL), which is associated with the phenomena of hemophagocytosis observed in the histological study and suggested HLH.Subsequently, the patient developed more pronounced pancytopenia , which included descending doses of dexamethasone and etoposide. Lumbar puncture was performed showing no disease, so triple intrathecal therapy was excluded from the treatment.In order to control EBV infection as a possible underlying trigger of HLH, monoclonal antibody Rituximab was added to the treatment. After starting Rituximab on 15 March 2022, the patient’s EBV viremia was negative and remains negative to date .Finally, for the treatment of HLH, the ABVD regimen  was decided upon for six cycles.During treatment, the patient presented multiple clinical complications, mainly hematologic toxicity and cytomegalovirus infection, which caused esophageal ulcers  leading Currently, the patient is being monitored and remains in complete remission with all secondary complications resolved with a current follow-up of 27 months. As previously described, we present the case of a patient with primary HIV and EBV co-infection. One of the elements that makes this a particular case is the exclusive involvement of the bone marrow by an HL. The secondary development of HLH made this a challenging and complex case to manage. Despite a broad differential diagnosis for esophageal stenosis being proposed, it finally turned out to be CMV involvement. Although the response rate for the treatment of HLH is variable, in many cases it causes long-lasting sequelae or high mortality . In our Regarding the initial presentation, the clinical findings, pancytopenia, and both analytical and CT imaging findings made it essential to perform a BM workup to reach a definitive diagnosis. In patients with HL and HIV infection, extensive extranodal involvement is frequent, including BM infiltration, although exclusive involvement of the BM is infrequent. This fact is probably explained by the underdiagnosis of this entity .Indeed, the fact that HL is not an AIDS-defining disease is an issue that will probably have to be reviewed in the future, since its incidence in immunocompromised populations, such as HIV patients, is high .On another note, as part of the diagnosis, a lymph node biopsy was performed which, although the incidence of lymph node involvement in this population is high, the histological findings were compatible with related lymphoid hyperplasia.The reasons for this conclusion were that these findings in an inguinal lymph node biopsy can occur in diseases other than HL, such as peripheral B- and T-cell lymphomas, infectious mononucleosis EBV, and follicular hyperplasia .Importantly, the prevalence of EBV infection globally is estimated to be around 90%, contributing to Hodgkin’s/Reed–Sternberg cell survival (HRS) .This circumstance, added to the high viral load of EBV demonstrated by PCR in the peripheral blood, made us consider related lymphoid hyperplasia as a diagnosis and not another cause contemplated in the differential diagnosis ,8,9.Finally, with respect to diagnostic features, the diagnosis of HLH is by exclusion. It is a life-threatening entity that must be considered in patients with cytopenias, increased transaminases, triglycerides and ferritin, coagulopathy, elevated IL-2 receptor, and decreased or absent NK cells, as well as the appearance of fever and splenomegaly . The preRegarding the secondary causes that can trigger this condition, autoimmune diseases, infections, malignant diseases, and drugs, among others, may be involved .In the presented patient’s clinical situation, HIV infection, EVB infection, HL, or a combination of the above could be causing the current clinical status.Although the criteria for HLH may be debated, the clinical presentation was compatible with this pathology, which contributed to the decision to start the HLH-94 protocol  with desAs in some other cases described in the literature, the coincidence of HL in the bone marrow together with the development of HLH made this case difficult to manage and diagnose, since the symptoms may overlap with each other, making interpretation difficult ,14.Regarding therapy and referring to HL, the treatment of advanced HL, defined as the accumulation of different criteria of a poor prognosis such as age over 45 years, stage IV, decreased serum albumin levels, male sex, anemia, and lymphocytosis or lymphopenia, is based on intensive BEACCOP type treatments . This is the recommended regimen for fit patients .The rationale for choosing a less intensive ABVD-type regimen was based on the decision of the multidisciplinary lymphoma committee not to increase the added toxicity to the regimen for the treatment of HLH .In addition, in patients with HL associated with lymphoproliferative neoplasms, initiation of HAART therapy is indicated .Despite HAART, incidence of HL has not decreased since its implementation and this could be explained in part by the dependence of HRS on CD4+ T lymphocytes .Chronic antigenic stimulation and co-infection with other oncogenic viruses appear to be key factors for lymphomagenesis in HIV patients. This may be the reason why even in the HAART era the incidence of neoplasms is higher in patients living with HIV than in the general population .Finally, in order to control EBV infection as a possible underlying trigger of HLH, monoclonal antibody Rituximab was added to the treatment as it is thought that it can eliminate EBV-infected B cells .As a consequence of all the treatments administered, the patient presented retrosternal pain and dysphagia. A gastroscopy was performed, and the biopsy was compatible with CMV infection. In addition, the patient developed cytopenia that required transfusion support and the delay in the treatment to be resolved. Despite the above, with a follow-up of almost two and a half years, the patient remains alive and in complete remission with symptoms derived from CMV esophagitis. Close ambulatory observation remains.The incidence of HL in the HIV population is increasing. Its presentation is mainly advanced, with exclusive medullary involvement being less frequent. EBV co-infection may play an important role in the etiopathogenesis of this type of lymphoma, with an increased incidence even in the HAART era. The three previous entities can produce HLH, a potentially fatal entity that requires specific treatment for its management. Multidisciplinary patient cooperation is fundamental for the treatment of these entities.


Parafilaroides spp. nematodes is an underreported disease in beached South American fur seals, with scant literature available on the characteristics of parafilaroidiasis, the nematode itself, as well as its occurrence in pinnipeds in Brazil. The present work aims to identify, describe and detail the histological features of the infection and molecular characteristics of verminotic pneumonia in the South American fur seal. Twenty-six specimens of Arctocephalus australis, found dead on the northern coast of Rio Grande do Sul in 2021, were analysed. These animals were identified and submitted to necropsy and histology. For the molecular identification of metastrongylids, lung fragments were subjected to DNA extraction, polymerase chain reaction targeting the Internal transcribed spacer 2 (ITS-2) gene and subsequent sequencing. In total, 12 animals presented with parasites in the lung parenchyma on histological evaluation, and only 1 showed a granulomatous lung lesion at necropsy. Microscopically, the nematodes were found mainly in the alveoli, associated with little or no inflammatory response, and they had morphological characteristics compatible with metastrongylids. Six ITS-2 gene quality sequences were obtained; after comparative analysis via BLAST, they showed similarity with sequences obtained from Parafilaroides sp. Therefore, verminotic pneumonia caused by Parafilaroides represents an important differential diagnosis of lung disease in South American fur seals found on the northern coast of Rio Grande do Sul.Verminotic pneumonia caused by  Parafilaroides spp. (Metastrongyloidea: Filaroididae) and Otostrongylus circumlitus (Metastrongyloidea: Crenosomatidae) is frequently reported in pinnipeds in different parts of the world    and fur seals  and the end of Palmares do Sul (30°28′40.4″S 50°19′18.9″W) in partnership with Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis . Twenty-six South American fur seals classified as codes 2 and 3, according to the decomposition classification, were identified and analysed as per the protocol of Geraci and Lounsbury . The age® Genomic DNA Mini Kit , according to the manufacturer's recommendations. For the PCR, primers (5′GCAGACGCTTAGAGTGGTGAAA3′/R 5′ACTCGCCGTTACTAAGGGAATC3′) flanked in the adjacent genes 5.8S and 28S were used, targeting the ITS-2 gene according to Lenerth et al. , as per the manufacturer's recommendations and sequenced on an automated sequencer . The generated sequences were submitted to BLAST® analysis  available from GenBank® using Clustal/W v.1.8.1  and the polyclonal anti-Brucella abortus primary antibody  produced in rabbits. Antigen retrieval was achieved with Proteinase K  for 10 min and the reactions were revealed with chromogen 3-amino-9-ethylcarbazole . As a positive control, lung slides from a bovine fetus infected with B. abortus  were classified as sub-adults, 8 as juveniles (30.8%) and 4 as adults (15.4%). Fifteen animals were males (57.7%) and 11 were females (42.3%). At necropsy, poor body condition was observed in 11 (42.3%) South American fur seals, good in 8 (30.7%) and moderate in 7 animals (26.9%). Gross and histological changes were analysed and the cause of death was determined for each fur seal when possible. Of these, 9 (34.6%) were diagnosed with traumas of different natures according to the macroscopic characteristics of the lesions – blunt trauma, puncture-cutting and blunt-blunt trauma (most frequent); 8 animals with poor body condition score and an empty stomach, sometimes containing liquid, were diagnosed with starvation (30.7%); and 9 were inconclusive (34.6%) due to unspecific morphological alterations  presented pulmonary alterations in all lung lobes, characterized by a moderate amount of circumscribed, white and sub-pleural nodular areas, with slight elevation, ranging from 0.5 to 2.5 cm in diameter, in addition to moderate diffuse oedema and multifocal to coalescing emphysema  of the South American fur seals, transverse and longitudinal sections of nematodes were observed, ranging from 30 to 130 e system . They wee system  and a ute system .Fig. 2.The parasites were predominately found in the alveoli, comprising 75% (9/12) of the fur seals that were positive on microscopy. They were mostly seen in the periphery of the lung lobes, close to the visceral pleura  or, less commonly, with a random multifocal distribution. Of the 9 cases in which parasites were seen in the alveoli, 5 were found exclusively in the alveolar lumen, while in 4 cases, there were also parasitic cuts in the bronchi or bronchioles. In 16.7% (2/12) of cases, parasites were seen in the bronchial space, and in 41.7% (5/12) they were identified in the bronchiolar space. Numerous emphysematous areas of variable extent, generally related to the number of nematodes , were asITS-2, 9 had bands of the expected size. The 6 sequenced samples  showed sequences compatible with Parafilaroides sp. registered in GenBank with accession numbers KP402084 and KP402085, by Jacobus et al. .The 6 individuals with confirmed parafilaroidiasis at PCR and histology had their lungs submitted for an immunohistochemical test to detect anti-Parafilaroides sp. in the South American fur seals from the present study was established by histological analysis, PCR and sequencing. At necropsy, Parafilaroides spp. are described as small and delicate parasites (Jacobus et al., et al., Parafilaroides spp. (Dailey, et al., The diagnosis of O. circumlitus and Parafilaroides spp., are mainly distinguished based on the characteristics inherent to their respective families, in which Crematosotidae has a bursa, while in Filaroididae, it is absent or indistinct, observed by the parasitic morphology that unfortunately we were unable to perform due to the absence of adult worms for analysis. Otostrongylus circumlitus is larger and therefore easily identified on macroscopy, in addition to being located in the bronchi, bronchioles and occasionally in the arteries. On the other hand, in Parafilaroides spp., it is smaller and predominantly alveolar in location (Onderka, et al., Otostrongylus circumlitus presents with an approximately 900 μm, with thick cuticle and hypodermis (Barnett et al., Parafilaroides spp., as seen in the present work, they have a diameter ranging from 30 to 130 μm, and a smooth and thin cuticle (Garner et al., The localization and histological characterization of the identified pulmonary parasites was compatible with the Metastrongyloidea Superfamily Bowman, . The 2 pParafilaroides spp. cause respiratory disease in pinnipeds and can result in high mortality rates (Dailey, et al., et al., Parafilaroides sp. died from traumatic causes, and 1 from starvation. These frequent causes of death in South American fur seals were also reported by other authors (Seguel et al., et al., et al., et al., et al., et al. (Sarcocystis sp. and gammaherpesviruses, had pneumonia caused by Parafilaroides sp. and was suggested as the cause of death, determined by the most severe lesion., et al.  which, dParafilaroides spp. and these are affected by other aetiological agents. It is further suggested that this nematode is a host for Brucella pinnipedialis (Rhyan et al., et al., B. abortus polyclonal antibody (Rhyan et al., Studies often describe pinnipeds parasitized by Parafilaroides have higher occurrences in certain regions, according to the literature already described (Measures, et al., P. normani (Dailey, et al., et al., et al., et al., P. normani.Apparently, the species of Parafilaroides, the phylogenetic analysis results showed high similarity with the sequences KP402084 and KP402085 with respect to ITS-2, which was deposited as Parafilaroides sp. by Jacobus et al. (ITS-2 sequences as Parafilaroides sp. when they sequenced a fragment of the cytochrome c oxidase subunit I mitochondrial DNA gene from one of their samples, it showed 100% similarity with P. normani (Jacobus et al., et al. (P. normani. Furthermore, the monophyletic relationship of the sequences from the present study with the sequences produced by Jacobus et al. (ITS-2 gene were satisfactory for sequencing and phylogenetic analysis.In addition to the fact that the morphology observed in the histological analyses was consistent with the genus s et al. . Althoug, et al. , as demos et al. , as wellParafilaroides spp. were evidenced in 1 animal, which presented with a nodular, multifocal and moderate lesion due to the formation of granulomas associated with the degenerated parasite. The histology results established the diagnosis, characterized the lung lesion and determined its intensity. Furthermore, PCR, with the amplification of a fragment of the ITS-2 gene, confirmed that the genus was involved in verminotic pneumonia. Combined, these approaches made it possible to suggest that the species involved in this infection is P. normani.Metastrongylids verminotic pneumonia was morphologically observed in the studied South American fur seals, and the lesions featured a mild-to-moderate morphological pattern. The characteristic macroscopy results of the lesion by


Autonomic innervation is important to regulate homeostasis in every organ of the body. The sympathetic nervous system controls several organs associated with metabolism and reproduction, including adipose tissue, the liver, and the ovaries. The sympathetic nervous system is controlled within the central nervous system by neurons located in the hypothalamus, which in turn are regulated by hormones like leptin. Leptin action in the hypothalamus leads to increased sympathetic activity in the adipose tissue. In this short report, we propose that leptin action in the brain also controls the sympathetic innervation of other organs like the liver and the ovary. We performed two experiments: We performed an intracerebroventricular (ICV) injection of leptin and measured norepinephrine levels in several organs, and we used a validated model of overnutrition and obesity to evaluate whether an increase in leptin levels coexists with high levels of norepinephrine in the liver and ovaries. Norepinephrine was measured by ELISA in adipose tissue and by HPLC-EC in other tissues. Leptin was measured by ELISA. We found that the ICV injection of leptin increases norepinephrine levels in several organs, including the liver and ovaries. Also, we found that diet-induced obesity leads to an increase in leptin levels while inducing an increase in norepinephrine levels in the liver and ovaries. Finally, since hyperactivity of the sympathetic nervous system is observed both in non-alcoholic fatty liver disease and polycystic ovary syndrome, we think that an increase in norepinephrine levels induced by hyperleptinemia could be involved in the pathogenesis of both diseases. In recent years, a wealth of evidence has emerged to support the close interplay between metabolism and reproduction. For example, hormones associated with metabolism and the body’s energy balance, such as leptin, glucagon-like peptide-1 (GLP-1), ghrelin, and insulin, regulate the secretion of gonadotropin-releasing hormone (GnRH) and, consequently, luteinizing hormone (LH) secretion and reproduction ,3. MetabLeptin is a peripheral hormone, released mostly from white adipose tissue (WAT), whose blood levels are proportional to the amount of body fat stored . The maiIn addition, leptin has been recognized to play a fundamental role in reproduction due to its role in puberty, controlling ovarian function  and moduBoth the ovaries and the liver are innervated by postganglionic sympathetic nerves originating from the celiac ganglion ,16. WithIn this short report, we aimed to unravel whether subchronic central leptin administration increases sympathetic innervation, using NE content in peripheral tissues as an indicator. We sought to first observe the NE content in different tissues in which SNS function has been well established, including the ovaries and liver. Furthermore, since bodily fat stores of WAT are known to differ in composition, we measured sympathetic NE content differed in different WAT stores in response to a central administration of leptin. Finally, because both the ovaries and the liver are innervated by sympathetic fibers ,16, we ap = 0.0570). In First, we measured NE concentrations in interscapular BAT (iBAT), subcutaneous WAT (scWAT), retroperitoneal WAT (rpWAT), and mesenteric WAT (mWAT) after 5 days of ICV leptin injection. The rationale for using a repeated dose of leptin in a short time was to simulate a sub-chronic increase in the hormone without generating leptin resistance. We observed an increase in NE concentration in iBAT and mWAT, while no changes were observed in rpWAT and scWAT A. AdditiWe employed an obesity model initiated during infancy, involving a reduction in litter size at birth, followed by the consumption of a high-fat diet (HFD) from weaning onwards . Control animals originated from normal litters and were provided a standard diet after weaning . Subsequently, animals were euthanized at postnatal day 50 (PND50) and postnatal day 150 (PND150). SL-HFD female rats showed higher levels of leptin at PND50 (young adulthood) and PND150 (adulthood) compared with the NL-SD A. NorepiWeight excess and adipose tissue dysfunction are involved in the pathogenesis of several diseases. Multiple mechanisms have been proposed as a link between obesity and the development of chronic diseases, including chronic low-grade inflammation ,21. ObesCentral injection of leptin for 5 days led to a simultaneous increase in NE levels in multiple tissues, including iBAT and WAT depots. Interestingly, when dissecting different WAT depots, NE levels increased only in mWAT following ICV leptin administration. This could imply a selective control of leptin in managing visceral fat depot stores. The reason for this specific impact of ICV leptin injection on iBAT and mWAT remains uncertain. However, it is established that various stimuli can differentially activate distinct white adipose tissue (WAT) depots . Indeed,Additionally, a central injection of leptin also led to a simultaneous increase in NE levels in the hypothalamus, liver, and ovaries. The SNS innervates the ovaries, and it is required for an adequate control of follicular development, steroidogenesis, and ovulation . In factSince early-onset obesity produces chronic hyperleptinemia, we used SL-HFD rats to test if the elevation of endogenous leptin levels linked to obesity also increases NE content in mWAT, the ovaries, and the liver. As expected, serum leptin was increased in SL-HFD rats at PND50  and PND150 . Moreover, NE levels in mWAT, the liver, and the ovaries increased at PND50 in SL-HFD rats compared to NL-SD rats. Similar results were observed at PND150, except for liver NE levels, which decreased. Interestingly, the liver is more susceptible to metabolic challenges. Indeed, a previous report demonstrated that 20 weeks of HFD consumption leads to neuropathy and loss of sympathetic innervation in the liver , which iWistar rats were bred and maintained in the dependencies of the vivarium of the University of Cordoba, Spain. All rats remained in a temperature-controlled room (22 ± 2 °C), in a 12 h light and 12 h dark cycle (light on at 08:00 a.m.), with access to food and water ad libitum. Vaginal smears were monitored each day, and only animals showing two normal estrous cycles were used. The animal protocols included in this study were approved by the Ethical Committee of the University of Córdoba; all experiments were conducted in accordance with European Union (EU) standards for the use and care of experimental animals . All efExperiment 1:We performed two experiments, as follows: We placed a cannula in the lateral ventricle of the brain to allow intracerebro-ventricular (ICV) delivery in nine adult female rats. Surgery was performed under ketamine/xylazine anesthesia. The cannulae were inserted at a point that was 1 mm posterior and 1.2 mm lateral to the bregma and were lowered to a depth of 3 mm beneath the skull surface. Weight and food intake were measured daily for 21 days after surgery. After 16 days, recombinant murine leptin  or aCSF were infused into the cannula twice a day for 5 days. Then, rats were euthanized by decapitation to collect interscapular BAT (iBAT), subcutaneous adipose tissue (scWAT), retroperitoneal adipose tissue (rpWAT), and visceral mesenteric adipose tissue (mWAT), as well as the liver, kidney, and ovaries. When rats were decapitated, we dissected the hypothalamus by making a horizontal cut approximately 2 mm in depth, guided by specific anatomical landmarks described in ,45. ThesExperiment 2:We used a previously validated model of overnutrition in which litters are reduced to 4 pups  and then, from weaning onwards, they are fed with a high-fat (HF) diet  ,47,48,49g at 4 °C for 15 min. The supernatant was collected and stored at −80 °C until the day of the test. Prior to the assay, an aliquot of 100 μL of the supernatant of the homogenates was mixed with 100 μL of 0.2 N PCA and filtered in 13 mm disposable filters of PVDF with a pore size of 0.22 μm (Millex™). Then, 20 μL of the filtrate was injected into the Jasco PU-2089s plus HPLC system coupled to the Jasco LC-NetII/ADC digitizer with a Kromasil 100-3.5-C18 column . To generate the integration of the chromatograms, the JASCO ChromPass Chromatography Data System v1.7.403.1 software was used. The mobile phase consisted of a buffer containing 0.1 M NaH2PO4, 0.14 mM octyl sulfate, 0.02% EDTA, and 1.5% acetonitrile, with a pH of 2.6. The flow rate was set to 1 mL/min. Under these conditions, the retention time was 5 min for NA. The potential of the amperometric detector was set at +750 mV.For the fat adipose tissue , NE concentration was measured using a norepinephrine ELISA kit . For the rest of the tissues, NE was quantified by high-performance liquid chromatography–coupled electrochemical detection . In brief, tissues were weighed in an analytical balance and homogenized manually in a glass–glass homogenizer in 200 µL of 0.2 M perchloric acid (PCA). The samples were centrifuged at 13,500× Taken together, in this brief report, we have documented that in female rats, leptin action in the brain increases sympathetic nerve content of NE simultaneously in iBAT and mWAT, as well as the hypothalamus, kidney, ovaries, and liver. This is likely a reflection of an increase in sympathetic tone and activity in those tissues, leading to dysfunction. The activation of sympathetic nerves could be a key component in the pathogenesis of several diseases associated with obesity and hyperleptinemia, including NAFLD and PCOS.An increase in norepinephrine concentration in the tissues could lead to different interpretations. Norepinephrine is mainly stored inside presynaptic vesicles of sympathetic fibers; thus, a high norepinephrine concentration could mean (1) an increased synthesis of norepinephrine, leading to more norepinephrine inside each vesicle, and (2) a high density of sympathetic nerves, in which the ramification of sympathetic nerves is increased. We did not measure the activity of sympathetic nerves, but both in obesity  and in P