In patients with arrhythmia, spiral real-time CMR yielded superior image quality to the conventional segmented imaging, allowing for excellent agreement with the reference echocardiographic volumetry. In contrast, in this cohort, standard Cartesian CMR showed significant underestimation of LV-ESV  and overestimation of LVEF ; (4) Conclusions: Real-time spiral CMR improves image quality in arrhythmic patients, allowing reliable assessment of LV volumetry.(1) Background: Segmented Cartesian Cardiovascular magnetic resonance (CMR) often fails to deliver robust assessment of cardiac function in patients with arrhythmia. We aimed to assess the performance of a tiny golden-angle spiral real-time CMR sequence at 1.5 T for left-ventricular (LV) volumetry in patients with irregular heart rhythm; (2) Methods: We validated the real-time sequence against the standard breath-hold segmented Cartesian sequence in 32 patients, of whom 11 presented with arrhythmia. End-diastolic volume (EDV), end-systolic volume (ESV), stroke volume (SV), and ejection fraction (EF) were assessed. In arrhythmic patients, real-time and standard Cartesian acquisitions were compared against a reference echocardiographic modality; (3) Results: In patients with sinus rhythm, good agreements and correlations were found between the segmented and real-time methods, with only minor, non-significant underestimation of EDV for the real-time sequence (135.95 ± 30 mL vs. 137.15 ± 31,  Cardiovascular magnetic resonance (CMR) is a well-established method for functional cardiac assessment and plays a pivotal role in modern diagnosis and clinical management of cardiovascular diseases ,2. HowevIn this study, we investigated the application of a tiny golden-angle b-SSFP spiral sequence for real-time cardiac imaging with the objective to validate its performance in direct comparison with the breath-hold segmented standard sequence for left-ventricular (LV) volumetry in patients with arrhythmias.This prospective study enrolled ambulant or hospitalized patients referred to CMR examination in our institution for various clinical indications from March 2019 to September 2021 and who consented for additional research scans. The study included 32 consecutive patients, of whom 21 had sinus rhythm. Eleven patients had arrhythmia in form of atrial fibrillation (nine patients) and ventricular extrasystoles (two patients) confirmed by electrocardiogram (ECG). Patients who had cardiac pacemakers, defibrillators, non-MRI compatible implants, or claustrophobia at the time of scanning were excluded.The study protocol was approved by the ethics committee of the University of Ulm and all participants provided informed consent in accordance with Declaration of Helsinki.All CMR scans were performed on a clinical 1.5 T magnetic resonance scanner . Acquisitions were performed in supine position using a 32-channel phased-array cardiac coil , and a vector electrocardiographic system was used for cardiac gating. The investigation was part of the conventional CMR protocol, and the real-time sequences were additionally performed at the end of the standard acquisitions.A set of 2D b-SSFP cine acquisitions was performed in two, three-, and four-chamber orientations followed by a stack of short-axis 2D b-SSFP cine slices spanning the entire left ventricle from the base to the apex. These scans were obtained with two sequences in each patient: segmented Cartesian acquisition and real-time spiral acquisition. Slice orientation, thickness, and interslice were the same for each sequence.Segmented Cartesian acquisition: Standard cine CMR was performed using a multi-slice b-SSFP Cartesian sequence. Retrospective ECG-gating was used to cover the entire cardiac cycle with 32 phases, leading to a temporal resolution of ~30 ms, depending on the heart rate. Contiguous slices were acquired in the short axis to allow full coverage of the ventricle . Each slice was acquired in ~4 ± 0.69 s (3 slices for 1 breath hold). CMR images were acquired during breath-holding in end-expiration. Detailed sequence parameters are presented in 3. A rewinder gradient was applied at the end of the spiral readout to maintain b-SSFP coherence. For each slice, data were acquired continuously for 3 s during free breathing over at least two R–R intervals, with the first R–R interval used to reach steady-state condition and the second for the actual image data acquisition. Data were reconstructed from seven consecutively acquired spiral readouts, yielding a real temporal resolution of 39.5 ms. Depending on the required number of slices for full LV coverage, the real-time sequences required an additional scan time of 44 s (11 slices)–68 s (17 slices). To address aliasing artifacts introduced by the 5-fold undersampling, all data were reconstructed offline by a k–t SPARSE–SENSE framework with a Total Variation (TV) sparsity operator, according to Wundrak et al. [Real-time spiral CMR acquisition and reconstruction: Real-time b-SSFP imaging was performed using a modified uniform density Archimedean spiral sequence, as described by Wundrak et al. , with a k et al. , and impTwo experienced cardiologists with board certification in cardiovascular MRI performed initial qualitative assessment of cine images before further quantitative assessment. A standardized assessment of image quality was performed according to the European CMR registry criteria . Higher The quantitative assessment of left-ventricular ejection fraction (LVEF), end-systolic volume (ESV), end-diastolic volume (EDV), and stroke volume (SV) was performed for each patient by one certified observer using the Segment software . For consistency and in accordance with recommendations of the Society for Cardiovascular Magnetic Resonance, the basal ventricular slice was defined as the plane in which 50% of the blood pool was surrounded by myocardium, and the most apical slice was determined as the last plane having blood pool at the end of diastole. The papillary muscles were not included in the calculation of LV mass quantification and were allocated to the ventricular cavity.In patients with arrhythmia, both segmented and real-time-based CMR volumetry were additionally compared to echocardiography as a reference gold-standard volumetry method. In these 11 arrhythmic patients, quantitative assessment of ESV, EDV with derived LVEF, and SV was performed according to well-defined standards by a cardiologist certified in transthoracic echocardiography by the European Association of Cardiovascular Imaging. Volumetry measurements followed the biplane method of discs (modified Simpson’s rule), using apical four-chamber and two-chamber views. As for CMR and according to the recommendations of the European Society of Echocardiography, the papillary muscles and trabeculations were assigned to the LV cavity. Measurements were performed using TomTec 4DLV analysis software 2.7 .Statistical analyses were performed using IBM SPSS software , STATGRAPHICS Centurion , and G*Power .Normality of the data was tested using the Shapiro–Wilk test. LV functional parameters derived from segmented Cartesian CMR, real-time CMR, and echocardiography were compared using paired-samples t-test or non-parametric Wilcoxon matched-pairs test. Bland–Altman analysis was performed among patients with sinus rhythm to assess agreement of the quantitative functional parameters measured by standard segmented method and spiral real-time method. The segmented data were used as the reference standard for Bland–Altman analysis. Proportional bias of volumetric variables was further evaluated using linear regression analysis. Inter-observer variability for the qualitative image scoring was assessed using a two-way intraclass correlation coefficient (ICC).Within the arrhythmic subgroup, additional analysis was performed to assess correlation, agreement, and equivalence between either the spiral or segmented method and the echocardiographic method defined as the reference standard for patients with arrhythmia. Correlation coefficients calculated using Pearson and Spearman methods were further supported by curve-estimation regression models. Additional two-sample equivalence tests were performed among arrhythmic patients to determine whether the means of either the segmented method or spiral method were equivalent with the means assessed by the reference echocardiographic method. Equivalence was demonstrated by showing that the 95% confidence interval for the difference between the means is entirely within the chosen margins of equivalence. A post hoc power analysis was also performed using G*Power, in order to assess the achieved power, for those two-tailed tests which rejected the null hypothesis. The analysis was performed based on the significance level of 0.05, the sample size considered for those cases, the correlation between the groups, and based on the effect size, determined from the difference between the dependent means of the two groups and their standard deviation, for the considered matched pairs.p-value of 0.05 or less was considered statistically significant.All results are expressed as the mean ± standard deviation. A p-value < 0.001). Real-time and standard segmented data sets were successfully acquired in all patients.A total of 32 patients were consecutively included in the study. Eleven patients had arrhythmia. The mean scan time of segmented Cartesian CMR was significantly longer than that of real-time spiral CMR  using spiral acquisition. The Bland–Altman analysis for the LV showed good agreements between spiral real-time CMR and segmented CMR with no significant degree of directional measurement bias, as shown in Biventricular metrics measured using spiral real-time and segmented Cartesian imaging are shown in Preliminary qualitative assessment showed more artifact burden in standard Cartesian cine imaging compared to spiral real-time cine method in the arrhythmic population. Representative images are depicted in In arrhythmic patients, volumetry obtained with both segmented standard CMR and spiral real-time method were separately compared with echocardiographic volumetry defined as the reference standard.p-value = 0.026) and significant overestimation of LVEF . The post hoc power analysis performed for the cases in which the null hypothesis was rejected showed a statistical power higher than 98% , for the considered sample size of 11. The computed statistical power shows, therefore, a very high probability for the correct rejection of the null hypothesis, in both cases. A summary of the data obtained with segmented Cartesian CMR protocol and real-time spiral technique compared to the echocardiographic method is presented in Volumetry obtained using segmented Cartesian CMR showed significant underestimation of LV-ESV (2 = 1), ESV , EF , and SV , as shown in 2 = 0.58), ESV , EF , and SV  as shown in Moreover, linear regression showed very strong correlation and agreement between real-time spiral CMR and echocardiographic acquisitions in EDV , ESV , SV  or LVEF  by showing that the 95% confidence interval for the difference between the means is entirely within the margins of equivalence, for the “spiral vs. echo” case, as shown in Reliable LV volumetric assessment using standard Cartesian CMR remains a challenge in patients with underlying arrhythmia. In this work, we present a real-time acquisition technique based on spiral k-space sampling combined with compressed sensing. The main findings of this study were that: (i) real-time b-SSFP imaging using spiral trajectories was feasible; (ii) there was good agreement for quantification of biventricular volumetry between the spiral real-time and standard segmented technique in patients with sinus rhythm; (iii) in arrhythmic patients, segmented Cartesian CMR showed significant underestimation of LV-ESV and significant overestimation of LVEF when compared to a reference echocardiographic technique; (iv) real-time spiral volumetry yielded strong agreement with and equivalent relation to volumetry obtained with the reference echocardiographic acquisitions in patients with arrhythmia.Various applications of spiral k-space sampling in cardiac MRI have been presented in the past ,19,20. H2 versus 1.4 × 1.4 mm2 for the standard cartesian CMR protocol. However, given the good agreement between the volumetry achieved with spiral real-time and standard CMR, the spatial resolution of the real-time CMR sequence does not have clinical implications.Our study showed that retrospective ECG-gated real-time CMR based on a spiral acquisition technique at 1.5 T had strong agreement with a standard segmented Cartesian-based sequence for biventricular volumetric assessment in patients with sinus rhythm. As reported in other studies, the main limitation of real-time techniques based on non-Cartesian trajectories with b-SSFP readouts relates to poor temporal resolution due to off-resonance effects and longer repetition times ,22,23,24The main goal of this study was to assess the performance of the real-time spiral sequence in patients with arrhythmia, where standard segmented CMR often fails to deliver robust volumetry. Metrics obtained with both CMR methods were compared to those assessed by an echocardiographic reference method. This objective quantitative approach is one strength of the current study, since previous and scarce data rely only on subjective qualitative scoring with inherent observer bias ,27. On tThe consistency of cardiac motion is pivotal for diagnostic qualitative imaging in standard segmented CMR. Therefore, irregular heartbeats might severely compromise volumetric assessment. The effect of mis-triggering related to arrhythmia is however unlikely to influence real-time CMR imaging, since all data for one slice were generated within one R–R interval.Indeed, we confirm in the present study superior image quality of the real-time CMR in arrhythmic patients in comparison to standard segmented CMR, allowing for reliable endocardial delineation. Standard segmented CMR showed significant underestimation of LV-ESV and significant overestimation of LVEF when compared to the reference echocardiographic technique in patients with irregular heartbeats. These results reflect the unreliable endocardial contouring with standard Cartesian imaging in arrhythmia, since segmented Cartesian CMR usually generates systematic volumetric overestimation when compared with echocardiography. In contrast, spiral real-time CMR yielded equivalent results with the reference echocardiographic acquisitions, as only minor, statistically non-significant overestimation of EDV could be observed. Previous studies which compared standard segmented CMR volumetry with echocardiographic volumetry demonstrated good agreements between the two methods in patients with sinus rhythm, despite overestimation of metrics obtained with CMR ,29. HoweA limitation of this study was the small sample size in a single center; however, it is comparable to previous work and the proportion of arrhythmic patients is superior to other studies that focused on volumetric accuracy in clinical practice ,31. NeveIn conclusion, we validated a spiral real-time b-SSFP sequence at 1.5 T, which enabled the assessment of biventricular volumes with a high degree of accuracy in subjects with sinus rhythm. In patients with underlying arrhythmia, compared to the segmented Cartesian imaging, spiral real-time CMR yielded superior image quality and better agreement with the reference echocardiographic volumetry. Therefore, we consider that this real-time sequence could be successfully applied in the arrhythmic population.


Overall, engagement and compliance from the crowd-sourced hand hygiene observation program, Clean-In-Clean-Out (CICO), were similar between 2019 (96.6%) and 2020 (96.7%) despite fluctuations within 2020 that reflected our hospital’s coronavirus disease 2019 (COVID-19) experience. Shared responsibility and just-in-time reminders can allow manual hand hygiene observation models to be sustainable. Hand hygiene is a cornerstone of infection prevention and is routinely tracked in hospital settings. During the coronavirus disease 2019 (COVID-19) pandemic, the main focus has been on airborne and droplet transmission, but the importance of hand hygiene has also had renewed emphasis. Severe acute respiratory coronavirus virus 2 (SARS-CoV-2) can survive on human skin for ∼9 hours, but it is easily inactivated with an alcohol-based hand rub, reinforcing the role of hand hygiene in limiting the COVID-19 spread. By contrast, we sought to determine whether UNC Medical Center’s Clean-In-Clean-Out (CICO) program, a well-established albeit manual method for hand hygiene observations, was sustainable throughout a public health and healthcare crisis. We also sought to determine whether the COVID-19 pandemic had an effect on hand hygiene compliance by longitudinally examining key metrics of the CICO program.To date, evaluations of hand hygiene in hospital settings during COVID-19 have focused on automated monitoring systems which collect compliance data through badge readers and/or dispenser use, eliminating the need for staff to perform traditional hand hygiene observations. Briefly, CICO can be accessed on any device on the hospital network via a web browser in all care settings. All staff are invited to participate with a suggested 5 observations per month, and they are encouraged to provide real-time feedback (compliment or reminder) when performing observations.UNC Medical Center utilizes a crowd-sourced hand hygiene audit application, CICO, to track hand hygiene observations and compliance.During this evaluation, key hand hygiene metric data were queried from the CICO application regarding the following elements: (1) engagement, the absolute number of observations performed overall and per 1,000 patient days; (2) compliance, hand hygiene compliance percentage; and (3) accountability, the percentage of observations with feedback from January 2019–December 2020 across inpatient settings. Weekly counts of COVID-19 hospitalized patients were determined by the weekly average number of COVID-19 patients on the midnight census.We made several comparisons to assess changes in data across 3 distinct periods. (1) Calendar year 2019 (baseline) was compared to calendar year 2020. (2) Calendar year 2020 was stratified into 4 periods based on the changing COVID-19 experience at our facility as follows: January–February was considered a prepandemic phase, with no COVID-19 patients, high census, and heightened awareness of COVID-19; March–June was characterized by COVID-19 patients and decreased overall census; July–September experienced the first COVID-19 surge with a return to high census; October–December was marked by high, sustained COVID-19 admissions along with high overall census. (3) To assess seasonal variation in hand hygiene compliance, October–December 2020 was compared to that from October–December 2019. Similarities were assessed by overlapping confidence intervals. Results are reported as mean with 95% CI.The 95% confidence intervals (CIs) were calculated using the standard formula for 95% CI for each of the periods and were compared to the 2019 baseline.Overall, there were 97,429 observations in 2019 and 74,809 in 2020. Adjusting for the number of observations per 1,000 patient days showed no statistically significant difference between 2019  and 2020 . Notably, total observations decreased below baseline levels during both the July–September  and October–December periods .Hand hygiene observations followed the same trend (sharp increase followed by stabilization) as COVID-19 hospitalizations from March through July 2020, when the number of COVID-19 hospitalizations reached its first peak  and 2020  (Fig. Not only was the Clean-In-Clean-Out model sustainable through a public health and healthcare crisis, initial increases in engagement and compliance metrics were identified. Although not at statistically significant levels, observations and feedback increased with COVID-19 media attention, prior to cases occurring in North Carolina or at our facility. These increases may be a reflection of staff proactively engaged in COVID-19 infection prevention efforts. Feedback and compliance increased again when our facility began seeing COVID-19 patients, even in the absence of active reminders and campaigns from infection prevention staff. This trend was further demonstrated by the decrease in hand hygiene compliance from October through December 2020, when our facility had high, sustained COVID-19 admissions as well as a normal non–COVID-19 patient volume.A statistically significant increase in hand hygiene compliance was observed during one period, March–June 2020, which corresponded with the admission of the first COVID-19 patient at our facility, shelter-in-place orders community-wide, and low inpatient volumes. In July, patient volumes started to return to pre–COVID-19 levels while our facility also began treating a higher number of COVID-19 patients. In addition, the hospital started experiencing staff shortages. The decrease in engagement observed with higher patient volume and patient-to-staff ratios is further evidence that operating at high capacity is not beneficial for patient safety. CICO is a novel hand hygiene program and is subject to selection and measurement bias. However, CICO provides benefits of real-time feedback that directly affects practices.This study had several limitations. We evaluated hand hygiene at a single, large academic medical center, and these results may not be generalizable to other settings or hand hygiene programs. However, the results were similar to findings published on COVID-19 and automated hand-hygiene monitoring systems in which compliance increased with lower patient volume, especially early in the pandemic. found that hand hygiene compliance decreased when hand-hygiene data sharing stopped. Furthermore, the crowd-sourced component of CICO allows all staff to complete observations and provide real-time feedback as time permits rather than further burdening infection prevention staff or specific designees who were inundated with competing priorities.Hand hygiene compliance remained high, >90%. Key CICO metrics demonstrated that although a manual rather than automated monitoring tool requires extra effort from staff, it is still a viable model. Unlike automated systems, CICO provides the opportunity for just-in-time reminders. Stangerup et al In the midst of a global pandemic, sustainable hand hygiene programs remain foundational to the success of infection prevention.The COVID-19 pandemic has caused healthcare staff to juggle numerous responsibilities with quality improvement and patient safety projects often being deprioritized and/or delayed. In the midst of this environment, it was encouraging to see that this CICO manual hand hygiene program was still actively utilized at comparable levels to the pre-COVID-19 baseline and that overall compliance and feedback provided did not significantly decline. In addition, the sustainability and success of the CICO program allowed UNC Medical Center to replicate this model of shared responsibility for monitoring mask compliance in contrast to more labor-intensive methods.


Tumor-derived exosomes (TEX) are a subset of small extracellular vesicles (sEV) present in all body fluids of patients with cancer. In plasma of patients with metastatic melanoma, numbers of exosomes produced by melanoma cells called MTEX are elevated. To study the role of MTEX in melanoma progression, immunoaffinity-based separation of MTEX from total plasma exosomes was performed. The surface of MTEX was decorated by various checkpoint inhibitory proteins, and upon coincubation with immune recipient cells, MTEX suppressed anti-tumor functions of these cells. MTEX emerge as a major mechanism of immune suppression in melanoma and thus might play a role in promoting melanoma progression. Extracellular vesicles (EVs) are produced and released by all cells. However, stressed cells, including cancer cells, produce an excess of EVs, and plasma of cancer patients is enriched in circulating tumor cell-derived vesicles called TEX. The main function of EVs is intercellular communication, which involves the transfer of information between cells, especially cells distantly located from one another. EVs are heterogenous and vary broadly in size, cellular origin, biogenesis and molecular/genetic cargos they carry . TEX are+ effector T-cell.This commentary discusses the interactions of TEX derived from melanoma cells (MTEX) with immune cells and describes the consequences of MTEX-mediated transfer of information from the tumor to immune cells. The impact of the MTEX-T cell crosstalk is emphasized as an example of cancer-driven functional reprogramming that promotes melanoma progression by inducing dysfunction of immune cells, especially CD8For the study described here , the banMTEX isolation from total plasma exosomes was performed by immune capture using the melanoma cell-specific monoclonal Abs provided by Dr. Soldano Ferrone (Harvard U.) Anti-CSPG4 mAbs (clones 763.64 and 225.28) recognize an epitope of CSPG4  present on melanoma cells but not on any other non-malignant cells or tissues as determined by immunostaining and previously reported . The capture mAb (763.64) was biotinylated, and MTEX captured by the biotinylated mAb harvested on streptavidin-charged magnetic beads as described . The non-captured vesicles were recaptured using biotinylated anti-CD63 mAb and streptavidin beads. Both fractions, MTEX and non-MTEX, were studied by on-bead flow cytometry for their protein profiles , and the+, while MTEX were CD3 (−). When MTEX were added to vesicles obtained from HDs plasma in spiking experiments, immune capture recovered all CSPG4+ vesicles, while the non-captured fraction was CSPG4 (−).Immunocapture of MTEX with anti-CSPG4 mAb proved to be highly effective, largely due to its specificity for the melanoma cells/exosomes and its high binding avidity. As such, it has been repeatedly used in our studies of MTEX. Specificity of the immunocapture for MTEX was verified by demonstrating that MTEX isolated from various melanoma cell lines showed the presence of CSPPG4 on all sEVs albeit at various expression levels; only MTEX isolated from plasma were CSPG4+, while non-MTEX were CSPG4 (-); sEV obtained from plasma of HDs were CSPG4 (-) and only MTEX were highly enriched in melanoma-associated antigens (MAA), TYRP2, Melan A, Gp100, VLA4; only non-MTEX were CD3immunosuppressive proteins was significantly higher (p = 0.03) for MTEX (15.3) than for non-MTEX (11.7). The mean RFI score for immunostimulatory proteins was significantly lower (p = 0.002) for MTEX (8.8) than for non-MTEX (16.2). Both suppressive and stimulatory scores for non-MTEX were similar to those calculated for sEV of HDs. We also calculated the stimulatory/suppressor ratio for MTEX, which was significantly lower (p = 0.001) than the ratio for non-MTEX or for sEV of HDs at 0.6, 1.4 and 2.2, respectively. In aggregate, the quantitative flow cytometry data indicated that MTEX were significantly enriched in immunosuppressive surface proteins, while non-MTEX largely carried immunostimulatory surface proteins. This observation indicated that MTEX are more likely to mediate suppression of immune cells than non-MTEX or sEV from plasma of HDs.On-bead flow cytometry of MTEX and non-MTEX was performed as previously described  to evalu+ T cells, CSFE-based proliferation of CD8+ T cells, apoptosis of CD8+ T cells and NKG2D down-regulation on the surface of NK cells. Utilizing primary in vitro activated CD8+T cells or natural killer (NK) cells as vesicle recipient cells in co-incubation experiments, we compared inhibitory effects of MTEX and non-MTEX on the above listed immune cell functions. MTEX captured on beads were used in these assays, while non-MTEX and sEV of HDs were in solution. The presence of beads alone or beads coated with biotinylated CSPG4 capture mAbs did not interfere with the functional assays (data not shown). Immunoregulatory activities of MTEX, non-MTEX and sEV of HDs were evaluated in co-incubation experiments with human primary immune cells . The folCoincubation of the isolated vesicles with human primary T cells or NK cells led to significant inhibition of activation, proliferation and survival of the immune cells interacting with MTEX but not of cells coincubated with non-MTEX or sEV obtained from plasma of HDs. The data in + T cells measured at 6 h of coincubation with MTEX may be initiated by cell surface signaling events, which then translate into cellular alterations in T cells. Indeed, using RT-PCR we showed that MTEX, but not non-MTEX, induced downregulation of CD69 mRNA transcripts in CD8+ T cells following 6 h coincubation. Also, a 30 min coincubation of CD8+ T cells with MTEX induced translocation of the NF-κB subunit p65 to the nucleus of a T cells, confirming activation of the NF-κB pathway and surface signaling by MTEX, which leads to downregulation of the CD69 expression level. It has been reported that the normally pro-inflammatory NF-κB pathway leads to cellular apoptosis in cancer, where stress due to genetic, metabolic or environmental factors drives the cell damage responses  mAbs. MTEX induced down-regulation of NKG2D (a cytolysis activating antigen) expression levels on the surface of NK cells, and thus inhibited NK cell activity. Preliminary experiments in which we attempted to interfere with the inhibitory signaling of MTEX by preincubation with neutralizing Abs to surface proteins on the MTEX surface showed only a partial and never complete inhibition of apoptosis. Also, the correlations linking the observed functional inhibition with expression of any single inhibitory protein on the MTEX surface were not significant, except for apoptosis, which was only partly blocked with anti-Fas mAb. The experiments with neutralizing mAbs suggested that not any one but rather several simultaneously delivered receptor-ligand signals might be responsible for MTEX mediated apoptosis in activated human primary CD8+T cells , linking the high TEP levels with the enrichment in suppressive MTEX. Thus, apoptosis correlated with the MTEX/TEP ratio . The RFI scores for FasL and TRAIL in MTEX were significantly elevated relative to non-MTEX, accounting for high MTEX-driven apoptosis. The stim/supp ratio correlated positively with the immuno-stimulatory score . Unexpectedly, apoptosis mediated by non-MTEX was inversely correlated with the stim/supp ratio  and with the immunostimulatory score . Non-MTEX -induced proliferation of T cells positively correlated with their immunostimulatory score . In aggregate, these and other correlations linking immune activities of MTEX and non-MTEX with their phenotypic characteristics showed that: i) MTEX had superior immune suppressor activity vis a vis non-MTEX; ii) the stimulatory/inhibitory vesicle activities were dependent on the surface profile of immunoregulatory proteins in MTEX and non-MTEX; and iii) MTEX and non-MTEX abilities to alter functions of immune receptor cells depended on the stim/suppr protein ratios in these vesicles.Functional changes induced in recipient immune cells by MTEX and non-MTEX were correlated with the protein profiles of these vesicles. Total exosome protein (TEP) levels in plasma correlated with the MTEX immunosuppressive score . This was an unexpected observation, which suggests that non-MTEX might potentially serve as a correlate of disease progression in future studies. We also observed a significant inverse correlation between of the stim/supp ratio with disease stage . This suggests that the stim/supp ratio might be more informative about disease progression than individual regulatory protein expression in the vesicular profile. In this study, with the exception of an inverse correlation between disease status and PD-L1 expression levels in MTEX , no other regulatory proteins on the MTEX surface correlated with disease status or activity.The group of patients we evaluated consisted of 12 individuals  aged 32–82 years, previously treated with oncological therapies. Five of these patients had no evident disease (NED) and seven had progressive disease at the time of blood draw for this study. We attempted to explore associations of the observed characteristics of MTEX and non-MTEX with disease status or activity in this small cohort of melanoma patients. While the study was not powered for a formal correlative assessment, it provided several potentially important insights. While MTEX-mediated apoptosis of CD8in vitro and in vivo in tumor-bearing mice  reduced CD69 expression levels and initiated apoptosis in CD8+T-cell or reduced NKG2D expression in NK cells contributing to the attenuation of NK activity  coincubation of MTEX with activated effector (CD8activity . These r


B) and collapsin response mediator protein 5 (CRMP5), in a patient with underlying small-cell lung cancer. We will discuss the differences in the two antibody syndromes, their significance, and their management.Paraneoplastic immune-mediated disorders have been well described in the literature. However, it is still relatively rare. The incidence has increased over the past decade due largely to the discovery of more autoantibodies. With a better understanding of the pathophysiology of different autoantibodies and clinical phenotypes, we are often able to diagnose clinically some specific paraneoplastic autoimmune neurological syndromes. We may also predict the response to treatment based on the autoantibody class. We are presenting a very unusual case of two completely different paraneoplastic syndromes with two different autoantibodies, gamma-aminobutyric acid-B (GABA Paraneoplastic nervous system disorders are neurological disorders occurring as an indirect, often immune-mediated response to underlying cancer rather than direct invasion by the cancer cells. They are relatively rare, with an incidence of 0.89 per 100,000 person-years, a prevalence of 4.37 per 100,000, and roughly one in every 300 cancers .With the discovery of more autoantibodies associated with underlying cancer, we are able to understand more about the pathophysiology of immune-mediated paraneoplastic neurological syndromes -6. The fWe present a very rare case of a combination of two paraneoplastic syndromes in a patient with small-cell lung cancer. We will discuss the clinical presentation, autoantibodies, pathophysiology, and management in more detail.A 66-year-old woman with a 50-pack-year smoking history experienced an episode of left-sided visual disturbance confirmed to be optic neuritis two years before her presentation to our service. She went on to develop impaired balance, upper and lower extremity weakness, Lhermitte’s sign, urinary incontinence, burning paresthesia, and anesthesia of the S2-4 dermatomes. Her symptoms were progressive, and she became wheelchair dependent with minimal vision in her left eye. Lumbar puncture reviewed oligoclonal bands in her cerebrospinal fluid (CSF). The aquaporin-4 (AQP4) antibody was negative. Magnetic resonance imaging (MRI) brain showed an increased signal in the left optic nerve and no other abnormalities. MRI spine did not show any lesions. She was diagnosed with probable multiple sclerosis.One year before the presentation, she was diagnosed with small-cell lung cancer (SCLC), for which she received chemotherapy and prophylactic whole-brain radiation.At the time of her presentation to us, she had two months of new-onset intractable seizures and worsening short-term memory. Her seizures consisted of spells of unresponsiveness with postictal confusion and were refractory to two antiepileptic medications at adequate doses.The patient’s examination was notable for short-term memory and cognitive deficits  19/30 with 0/5 delayed recall). She had left optic disc pallor, a left afferent pupillary defect, and reduced visual acuity (20/40 on the right and 20/100 on the left). She was non-ambulatory, with 3/5 strength in the left leg and 4/5 in the right leg. Sensory testing revealed S2-4 anesthesia. Reflexes were 2+ throughout, with upgoing plantar responses.She had a normal interictal electroencephalogram (EEG). CSF showed the presence of oligoclonal bands and elevated IgG index with normal cell counts, protein, culture, and encephalitis panel. Aquaporin4 (AQP4 IgG) and anti-myelin-oligodendrocyte glycoprotein (MOG IgG) testing were negative. MRI showed T2 hyperintensity throughout the left hippocampus and right amygdala with atrophy of the right hippocampus; there were no contrast-enhancing lesions Figure .B) and collapsin response mediator protein 5 (CRMP5) antibodies at high titers. The patient was diagnosed with autoimmune limbic encephalitis secondary to anti-GABAB receptor antibodies. Her myelitis and optic neuritis were determined to be paraneoplastic in the setting of known SCLC and anti-CRMP5  antibodies. The patient was started on treatment with rituximab and subsequently showed significant improvement in her seizure control. However, her sensorimotor deficits remained, and she continued to have cognitive decline and occasional breakthrough seizures.A serum autoimmune encephalopathy panel was positive for both gamma-aminobutyric acid-B , leading to a B-cell-mediated immune response and inflammation. They are less consistently associated with underlying malignancy than intracellular antibodies. Treatment of cell surface antibody-mediated disease involves plasmapheresis or immunosuppression  in addition to treating any underlying tumors .B receptor antibody limbic encephalitisGABAB receptor antibody is a cell surface antibody primarily known to cause limbic encephalitis and is associated with an underlying neoplasm in about half of cases [B receptor antibody encephalitis has remained an uncommon condition, with a few hundred cases identified to date [B receptor antibodies often presents with subacute (days to months) seizures, cognitive decline, short-term memory deficits, and behavioral or psychiatric disturbance. Seizures in this form of limbic encephalitis are classically focal with impaired awareness and often resistant to standard anti-epileptic medications [The GABAof cases . The mosof cases . Since i to date . Cases hications ,12,13.CRMP5 antibody-associated myelitis, optic neuritisThe CRMP5 antibody is an intracellular antibody that is almost always paraneoplastic and most commonly seen in SCLC (about 75% of cases), again not with other types of lung cancer ,15. ClinIn the case of our patient, the combination of optic neuritis and presumed myelitis with CSF oligoclonal bands led to an initial diagnosis of probable multiple sclerosis (MS). Patients with atypical presentations of MS or neuromyelitis optica spectrum disorder (NMOSD) and significant risk factors for malignancy, autoimmune or paraneoplastic encephalitis should be considered early with a high degree of suspicion.The incidence and prevalence of autoimmune encephalitis have increased dramatically during the past decade due to the identification of novel autoantibodies. Many of these cases present as paraneoplastic syndromes, often preceding the final diagnosis of underlying cancer. Overall, these syndromes are still relatively rare. Of the two main types of autoantibodies, those against intracellular antigens are not directly pathogenic and instead serve as a disease marker for underlying malignancy. In contrast, autoantibodies against cell surface or synaptic antigens are directly pathogenic as they induce a B-cell-mediated autoimmune response.B paraneoplastic autoimmune limbic encephalitis after the cancer diagnosis. Our case also demonstrated the clinical features of the two paraneoplastic immune-mediated neurological disorders.Our patient is unusual due to the presence of both types of autoantibodies associated with underlying SCLC. The patient presented over a two-year period, first with CRMP5-associated paraneoplastic syndrome one year before the cancer diagnosis and later with a typical GABA


As the coronavirus disease 2019 (COVID-19) pandemic continues, there has been a growing interest in the chronic sequelae of COVID-19. Neuropsychiatric symptoms are observed in the acute phase of infection, but there is a need for accurate characterization of how these symptoms evolve over time. Additionally, African American populations have been disproportionately affected by the COVID-19 pandemic. The COVID-19 Neurological and Molecular Prospective Cohort Study in Georgia (CONGA) was established to investigate the severity and chronicity of these neurologic findings over the five-year period following infection.The CONGA study aims to recruit COVID-19 positive adult patients in Georgia, United States from both the inpatient and outpatient setting, with 50% being African American. This paper reports our preliminary results from the baseline visits of the first 200 patients recruited who were on average 125 days since having a positive COVID-19 test. The demographics, self-reported symptoms, comorbidities, and quantitative measures of depression, anxiety, smell, taste, and cognition were analyzed. Cognitive measures were compared to demographically matched controls. Blood and mononuclear cells were drawn and stored for future analysis.Fatigue was the most reported symptom in the study cohort (68.5%). Thirty percent of participants demonstrated hyposmia and 30% of participants demonstrated hypogeusia. Self-reported neurologic dysfunction did not correlate with dysfunction on quantitative neurologic testing. Additionally, self-reported symptoms and comorbidities were associated with depression and anxiety. The study cohort performed worse on cognitive measures compared to demographically matched controls, and African American patients scored lower compared to non-Hispanic White patients on all quantitative cognitive testing.Our results support the growing evidence that there are chronic neuropsychiatric symptoms following COVID-19 infection. Our results suggest that self-reported neurologic symptoms do not appear to correlate with associated quantitative dysfunction, emphasizing the importance of quantitative measurements in the complete assessment of deficits. Self-reported symptoms are associated with depression and anxiety. COVID-19 infection appears to be associated with worse performance on cognitive measures, though the disparity in score between African American patients and non-Hispanic White patients is likely largely due to psychosocial, physical health, and socioeconomic factors. •Neuropsychiatric symptoms are often reported following COVID-19 infection.•Self-reported symptoms may not be associated with objective dysfunction.•Self-reported symptoms may be associated with depression and anxiety.•Cognitive testing may overestimate clinical impairment in disadvantaged populations. PrelimiMuch of the current literature on the long-term neuropsychiatric sequelae of COVID-19 has relied on self-reported symptoms through questionnaires administered either in-person or through telephone interviews to comply with public health guidelines. However, the correlation between these self-reported symptoms and objective dysfunction is unclear. Self-reported depression has been demonstrated to overestimate clinical depression while self-reported symptoms such as anosmia and dysgeusia oftentimes underestimate the true prevalence of taste and smell dysfunction .There is an obvious need for accurate characterization of the neuropsychiatric sequelae of COVID-19 and the risk factors associated with these outcomes. Such research should be cognizant of the potential disparities in how COVID-19 affects different populations. African Americans are estimated to be 13% of the United States population, yet they are 2.8 times more likely to be hospitalized and twice as likely to die from COVID-19 compared to White, non-Hispanic persons according to the Centers for Disease Control and Prevention . As the 22.1This is a single center, prospective, closed cohort study. We recruited adult patients who had tested positive for COVID-19 infection by respiratory swab or saliva sample reverse transcriptase polymerase chain reaction (RT-PCR) from the Central Savannah River Area (CSRA) of Georgia, where Augusta University (AU) Health System is a statewide COVID-19 testing site and has performed the majority of the testing for symptomatic and non-symptomatic individuals in the communities of CSRA. Recruitment began in March 2020, initially by contacting patients with a positive test through the AU Health testing services through telephone, and subsequently including a flier which allowed patients who were not tested through AU health to participate provided they submitted documentation of positive COVID-19 infection via testing by respiratory swab RT-PCR or saliva RT-PCR. Patients were evaluated at the clinical trials office at AU following their diagnosis with the goal of annual evaluations until five years after their baseline visit. This manuscript follows the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines.2.2CONGA Cohort: Inclusion criteria included patients testing positive for COVID-19 infection by respiratory swab or saliva sample RT-PCR. Enrolled patients were a minimum of four weeks from the date of confirmed COVID-19 infection or four weeks after the patient was discharged from the hospital. Both hospitalized and non-hospitalized patients were included. Hospitalized patients were tested upon admission. Participants had to be residents of the state of Georgia and older than 18 years old. Individuals with cognitive impairment and impaired decision-making capacity were included if consent was provided on their behalf by a legally authorized representative.Patients were excluded if they were non-English speaking for whom a cognitive screen conducted in English would be an inaccurate representation of cognition.Patients who met inclusion criteria were recruited via telephone call using an approved telephone script abbreviating details of the study and via community outreach efforts in COVID-19 education.Control Cohort: The control cohort included participants who were enrolled in either the Georgia Cardiovascular Twin Study or the Georgia Stress and Heart study from 4/30/2019 to July 7, 2021. Criteria for classifying participants as African Americans or European Americans as well as study design and selection criteria have previously been described , highest level of education, and self-identified race . Participants also reported if they exercised, were hospitalized, had ever smoked tobacco products, were currently consuming alcohol, and had any medical comorbidities. The questionnaire also asked about symptoms attributed to acute COVID-19 infection with the option to write in other symptoms experienced. Patients also self-reported comorbid conditions with the option to write in any additional medical conditions.Clinical measures included a full neurological examination including mental status, cranial nerves, motor function, sensory function, reflexes, coordination, and gait. Cognitive assessments included a Montreal Cognitive Assessment (MoCA) and NIH Toolbox (NIH-TB) for the Assessment of Neurological and Behavioral Function studies. NIH-TB studies included the tests of working memory (List Sorting Working Memory Test) and language (Picture Vocabulary Test). Scores were reported as age-adjusted national percentiles. Cognitive impairment was defined as a MoCA score less than or equal to 26 which is the cutoff for mild cognitive impairment . NIH-TB A University of Pennsylvania Smell Identification Test (UPSIT) was to be completed at home. The UPSIT contains 40 multiple-choice questions involving the identification of microencapsulated odors to determine the degree of smell loss . A WaterSubjects completed mood questionnaires during their visit, including a Patient Health Questionnaire-9 (PHQ-9) for depression, which was defined as a score of 10 or higher, and a Generalized Anxiety Disorder-7 (GAD-7) screen for anxiety which was defined as a score of 8 or higher .2.4Descriptive statistics were provided for the demographic information and neuropsychiatric measures in our cohort. Spearman's rank correlation test was utilized to calculate the correlation among the neuropsychiatric measures. Correlation strength was defined as follows: 0.00–0.19 (very weak), 0.20–0.39 (weak), 0.40–0.59 (moderate), 0.60–0.79 (strong) and 0.80–1.00 (very strong) . Very we2.5This study protocol was approved by the Institutional Review Board (IRB) at Augusta University Medical Center. Written consent detailing the purpose, procedures, data storage, risks, and benefits of the study was obtained at the initial visit. Given that we expected to encounter subjects with cognitive deficits during our study, patients completed a modified 10-question University of San Diego Brief Assessment of Capacity to Consent (UBACC) prior to providing informed consent independently with the option of consent provided by a legally authorized representative otherwise .4To date, over 20,000 cases in the CSRA have tested positive through our institution. At the time of analysis, 200 participants had been assessed at our clinical trials office. The demographic characteristics of the 200 participants included 71 males [35.5%] and a mean age of 44.6 years [Range 19–82]) . 79 partThe prevalence of at least one self-reported neurological symptom attributed to COVID-19 was 160 out of 200. The most commonly reported COVID-19 symptom was fatigue (137 out of 200 patients [68.5%]), which was followed by headache (133 out of 200 [66.5%]) . 109 out4.149 out of the 196 patients (25%) who completed a PHQ-9 met the criteria for depression . 35 out Out of the 164 subjects that had returned the UPSIT at the time of analysis, 49 (29.9%) had clinical evidence of impaired olfaction. Out of the 159 subjects that had returned the WETT at the time of analysis, 47 (29.6%) had clinical evidence of impaired taste. Age, sex, and ethnicity were not significantly associated with smell or taste impairment.4.2A Spearman rank correlation was performed among the neuropsychiatric measurements . Strong 4.3There were no significant associations between self-reported hyposmia and hypogeusia attributed to COVID-19 and their associated quantitative measures. The percentage of patients with abnormal MoCA and NIH-TB scores did not significantly differ between patients endorsing cognitive symptoms compared to those who did not.4.4There were many significant associations between self-reported symptoms significantly associated with depression and anxiety . The sum4.5342 participants were enrolled in either the Georgia Cardiovascular Twin Study  or the Georgia Stress and Heart study (n = 112) from 4/30/2019 to July 7, 2021. The general characteristics of the control cohort and CONGA cohort patients who completed the NIH-TB tests are summarized . The aveCompared to the control cohort, the average percentile on the NIH-TB working memory test in our study was significantly lower in both the general cohort and in patients under the age of 50 . However, the percentage of patients with impaired working memory in the control cohort compared to the CONGA cohort was not statistically significant .African American participants performed worse in all cognitive tests compared to White, non-Hispanic participants after controlling for age, sex, and education .Table 8E55.1We analyzed the preliminary data from the baseline visits of the first 200 patients enrolled in the CONGA study. Novel findings include evidence that self-reported symptoms may not correlate with quantitative testing. These data underscore the importance of quantitative testing in the accurate assessment of deficits. African American patients appear to score significantly worse on quantitative cognitive testing compared to Non-Hispanic White patients, which likely underscore the disparities in how cognitive tests assess different ethnic groups due to various systemic factors including differences is socioeconomic status, psychosocial factors, and physical health. Findings of the present investigation reinforce the evidence from previous studies that patients who have been infected with COVID-19 continue to report neuropsychiatric effects beyond the acute phase of infection, which has been termed “long COVID” or PASC. We found evidence of worsened cognitive performance in our cohort compared to the control group though it did not meet our threshold for clinical significance.5.2Self-reported change in smell is an established predictor of acute COVID-19 infection . Yet, whIn fact, a higher proportion of clinical anosmia and dysgeusia was noted in patients who did not report a change in smell and taste. There are multiple explanations for why those who self-report olfactory and gustatory changes may not present with evidence of objective abnormalities on clinical testing. It is possible that participants recovered those senses following acute infection as the mean time from positive COVID-19 test to clinical visit in our study was 125 days. A recent review of olfactory dysfunction following COVID-19 infection found that resolution of self-reported symptoms occurs an average of 10 days after symptom onset . Additio“Brain fog” is a colloquial term utilized to describe the constellation of cognitive symptoms following COVID-19 infection and has been considered a significant and debilitating aspect of long-COVID . However5.3In the present investigation, 80% of patients reported neurological symptoms. The most common reported COVID-19 symptom was fatigue (137 out of 200 patients [68.5%]) followed closely by headache (133 out of 200 [66.5%]) which is similar to what has been previously reported . Patient5.4Compared to our control cohort, the CONGA cohort scored significantly lower on the NIH-TB working memory test based on age-adjusted national percentiles. Previous data published with NIH-TB testing in COVID-19 patients also found that working memory was impaired in COVID-19 patients compared to the general population based on demographic-matched normative scores . Notably5.5The prevalence of persistent objective smell and taste dysfunction (30% and 30% respectively) in our cohort is well within the range of what has been previously reported via subjective measures and quantitative olfactometry in the literature . Previou5.6With many patients reporting symptoms beyond the acute phase of COVID-19, there is a necessity for effective treatments. One intervention which has demonstrated effectiveness for psychiatric symptoms is cognitive behavioral therapy (CBT) . Specifi5.7Strengths of our study include the large sample size, the unique racial diversity of our community, and the use of validated quantitative measurements of smell, taste, and cognitive function. Limitations to this study include the inability to administer smell and taste tests in the clinic setting to follow public masking health guidelines resulting in a lower yield of returned test results. Given the high prevalence of COVID-19 during the pandemic, it is impossible to rule out exposure to COVID-19 in the control group, though we attempted to minimize this effect by excluding patients utilizing the screening questionnaire. Additionally, patients who seek out SARS-CoV-2 testing may have intrinsic differences than those that do not, such as the motivation and health literacy to seek out tests.5.8We feel that our study should be generalizable to non-hospitalized COVID-19 patients while understanding that geographic and demographic differences may affect outcomes. Given the relatively low percentage of hospitalized patients (7%), this study may not be representative of the outcomes of hospitalized patients.D.C.H. receives funding from the National Institute of Neurological Disorders and Stroke ). The remaining authors declare that they have no known competing financial interests or personal relationships that could appear to influence the work in this paper.10.13039/100000065National Institute of Neurological Disorders and Stroke. David Hess reports financial support was provided by TR Reddy Family Fund.David Hess reports financial support was provided by


What are the treatment outcomes and adverse events among pregnant patients with multidrug-resistant tuberculosis (MDR-TB)?In a systematic review and meta-analysis of 10 studies including 275 pregnant patients with MDR-TB, the pooled proportion of treatment success was 72.5%, and the pooled proportion of favorable pregnancy outcomes was 73.2%. Adverse events, such as liver function impairment, kidney function impairment, hearing loss, and hypokalemia, were common among pregnant patients with MDR-TB, occurring in more than half of the patients.This study suggests that a high rate of treatment success and favorable pregnancy outcomes can be achieved when pregnant patients with MDR-TB are treated with effective regimens. The management of multidrug-resistant tuberculosis (MDR-TB) during pregnancy is challenging, yet no systematic synthesis of evidence has accurately measured treatment outcomes.To systematically synthesize treatment outcomes and adverse events among pregnant patients with MDR-TB.PubMed, Scopus, Web of Science, and ProQuest were searched from the inception of each database through August 31, 2021.Studies containing cohorts of pregnant patients with a defined treatment outcome were eligible.Independent reviewers screened studies and assessed the risk of bias. The study followed the Preferring Reporting Items for Systematic Review and Meta-analyses reporting guideline. Meta-analysis was performed using random-effects models. The sources of heterogeneity were explored through metaregression.The primary outcome was the proportion of patients with each treatment outcome , and the secondary outcomes included the proportion of patients experiencing adverse events during pregnancy.In this systematic review and meta-analysis, 10 studies containing 275 pregnant patients with available data on treatment outcomes were included. The pooled estimate was 72.5%  for treatment success, 6.8%  for death, 18.4%  for loss to follow-up, and 0.6%  for treatment failure. Treatment success was significantly higher in studies in which the proportion of patients taking linezolid was greater than the median (20.1%) compared with studies in which this proportion was lower than the median . More than half of the pregnant patients  experienced at least 1 type of adverse event, most commonly liver function impairment , kidney function impairment , hypokalemia , hearing loss , gastrointestinal disorders , psychiatric disorders , or anemia . The pooled proportion of favorable pregnancy outcomes was 73.2% . The most common types of adverse pregnancy outcomes were preterm birth , pregnancy loss , low birth weight , and stillbirth . Most of the studies had low-quality (3 studies) or medium-quality (4 studies) scores.In this systematic review and meta-analysis, high treatment success and favorable pregnancy outcomes were reported among pregnant patients with MDR-TB. Further research is needed to design shorter, more effective, and safer treatment regimens for pregnant patients with MDR-TB. This systematic review and meta-analysis examines treatment outcomes and adverse events among pregnant patients with multidrug-resistant tuberculosis (MDR-TB). The physiological dynamics of pregnancy might affect pharmacokinetic parameters that could lead to inadequate treatment and poor outcomes.3 A recent systematic review showed that MDR-TB is associated with a high risk of adverse maternal and perinatal outcomes, such as maternal death, pregnancy loss, preterm birth, and low birth weight.4Multidrug-resistant tuberculosis (MDR-TB) is a major public health concern affecting approximately half a million people globally, but only 57% of cases of MDR-TB were successfully treated in 2019.5 However, the adverse effects of these drugs on pregnancy outcomes have yet to be comprehensively quantified. Moreover, the treatment outcomes among pregnant patients with MDR-TB have not been well studied, with only a few observational studies available. The findings from these studies have provided inconclusive evidence. Therefore, we conducted a systematic review and meta-analysis of the available evidence on treatment outcomes among pregnant patients with MDR-TB. Our primary objective was to quantify the pooled proportion of patients with each treatment outcome, including treatment success, death, loss to follow-up, and treatment failure. Our secondary objective was to assess adverse pregnancy outcomes and adverse drug events. In addition, we aimed to assess study characteristics and TB medicines that may be associated with outcomes, particularly treatment success.Although untreated MDR-TB is associated with significant maternal mortality, morbidity, and adverse pregnancy outcomes, there is no standardized regimen for treatment of MDR-TB among pregnant patients. There are currently limited options for second-line TB drugs for the treatment of pregnant patients with MDR-TB. World Health Organization (WHO) guidelines on the management of drug-resistant TB during pregnancy recommend the use of individualized, longer regimens containing at least 4 effective drugs with an established safety profile and low teratogenic risk.PRISMA) reporting guideline.6 The review protocol has been registered in PROSPERO (CRD42021273215).7This systematic review and meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses  independently screened the titles, abstracts, and full texts to identify eligible studies. Any discrepancies were discussed and resolved by consensus. EndNote, version X8 (Clarivate) and Rayyan (Rayyan)12 The tool has scores ranging from 0 to 9 with low-quality (0-4), medium-quality (5-7), and high-quality (8-9) groupings  using the Newcastle-Ottawa Scale.Q test and quantitatively measured by the index of heterogeneity squared (I2) statistics and corresponding 95% CIs.13 Heterogeneity was considered low when I2 values were below 25%, moderate when I2 values were between 25% and 75%, or high when I2 values were above 75%.13 The sources of heterogeneity were explored through metaregression using study characteristics as covariates. The WHO grouping of medicines recommended for use in longer MDR-TB regimens was also used as a covariate .Meta-analysis was performed using a random-effects model to estimate the pooled proportion of treatment outcomes, pregnancy outcomes, or adverse events. To account for the sample size differences between the studies, we conducted the meta-analysis using inverse variance weights. The inverse variance weight represented a composite measure of total variances and sample size such that studies with larger sample sizes were given more weight than smaller studies. This choice of weights minimizes the uncertainty of the pooled effect estimate. Analyses were performed separately for each of the outcomes when 2 or more studies were available on the outcome of interest. The summary effect estimates and 95% CIs for individual outcomes were represented with a forest plot. Heterogeneity between studies was examined using the Cochran 23 were included in the meta-analysis, which included 288 pregnant patients with MDR-TB .The studies were conducted in 5 different countries, and the data were collected from 1996 to 2019   . The pooled proportion of pregnant patients whose outcome was loss to follow-up was 18.4%  . Treatment success was significantly higher in studies with a higher percentage  of linezolid-containing regimens  than in studies with a lower percentage of linezolid-containing regimens . Treatment success rates did not differ by other TB medicines  of patients had no reported adverse events, whereas 54.7%  of patients experienced at least 1 type of adverse event (eTable 9 in the Drug-related adverse events were reported in 4 studies (86 patients)22 (eTable 10 in the Six studies reported on pregnancy outcomesP = .72) .33 Given the limited drug options available to treat individuals with MDR-TB, especially pregnant patients, the high proportion of patients with treatment success in our study compared with previously reported findings in the general adult population is encouraging.The overall pooled treatment success among pregnant patients with MDR-TB was 72.5%, which is close to the WHO target of 75% and lower than the findings from previous systematic reviews and meta-analyses on treatment success among children with MDR-TB (77.0%-83.4%).8 Our subgroup analysis found that the treatment success rate was significantly higher for studies with a higher percentage of linezolid-containing regimens (85.0%) than for studies with a lower percentage of linezolid-containing regimens (65.6%). However, this finding might be confounded by the use of other drugs, such as bedaquiline and delamanid, because the associations of these drugs with treatment outcomes were not assessed because of the small number of studies in which they were used. Previous systematic reviews and meta-analyses showed that linezolid was significantly associated with higher treatment success rates and lower mortality rates among adult patients with MDR-TB.34 All of these findings indicate that linezolid might be used to effectively treat pregnant patients with MDR-TB. However, a meta-analysis of individual participant data would be required to assess the independent association of each drug, including linezolid, with treatment success. Linezolid cannot be used for long periods because of hematologic adverse effects. Further research on the safety, tolerability, timing, and duration of linezolid, bedaquiline, and delamanid treatment during pregnancy, using adequately powered studies, is a priority.The use of recently recommended novel drugs—including bedaquiline, delamanid, and linezolid—for the treatment of MDR-TB during pregnancy may increase the treatment success rate and help to achieve the WHO targets.33 The loss to follow-up problem among pregnant patients could be mitigated with more integrated care because pregnant patients often access medical care during pregnancy. Our metaregression showed that the pooled proportion of loss to follow-up was significantly associated with high HIV prevalence and older age. This finding is consistent with previous studies reporting that HIV coinfection was associated with a high rate of loss to follow-up among patients with MDR-TB.35 This finding could occur because loss to follow-up includes unrecorded deaths. Adopting and implementing a shorter MDR-TB treatment regimen  recently recommended by the WHO might be a solution to reduce the high rate of loss to follow-up.8 A previous systematic review conducted to identify strategies for reducing loss to follow-up showed that engagement of community health workers as treatment professionals and the provision of patient education was associated with lower rates of loss to follow-up.36 Financial support and psychological counseling were also reported as important interventions associated with reducing the rate of loss to follow-up and treatment failure.8Although the pooled proportion of patients experiencing treatment failure was low (0.6%), the pooled proportion of loss to follow-up was high (18.4%). This finding suggests that loss to follow-up was a major challenge to achieving a high rate of treatment success among pregnant patients with MDR-TB, which is consistent with the rates of loss to follow-up among other populations with MDR-TB (12%-17%).33 More important, our metaregression indicated that mortality rates were higher in studies that include patients with both PTB and EPTB than in studies that include patients with only PTB. This finding could be due to late diagnosis and treatment because early diagnosis is a challenge for EPTB cases. Extrapulmonary TB can also be associated with other confounding risk factors for death, such as immunosuppression and chronic illness.37Our systematic review and meta-analysis also showed that the pooled proportion of patients with MDR-TB who died during treatment was 6.8%. This finding is consistent with results from previous systematic reviews on adults and children with MDR-TB.38 High rates of drug-associated adverse events in the treatment of patients with MDR-TB were also reported in a recent meta-analysis using individual patient data.39 The high frequency of adverse events suggests that pregnant patients receiving medication for MDR-TB should be monitored closely and managed aggressively for adverse effects. Data were not available in our systematic review to identify the type of adverse events associated with specific TB drugs.We found that adverse events were common among pregnant patients with MDR-TB, with more than half of the patients (54.7%) experiencing at least 1 type of drug-related adverse event. This finding is consistent with a previous systematic review that found that 57.3% of patients with MDR-TB experienced at least 1 kind of adverse event, with gastrointestinal disorders, ototoxicity, and psychiatric disorders being the most common.Although different treatment regimens were used by pregnant patients, the most common types of TB medicine included in the regimens were pyrazinamide, cycloserine, prothionamide, and fluoroquinolones. It was not possible to assess the independent associations of these drugs with treatment outcomes because various regimens were used for the treatment of MDR-TB. Although some second-line TB medications, such as amikacin, streptomycin, prothionamide, and ethionamide, are considered teratogenic and are usually contraindicated for pregnant patients, these drugs were used in some of the included studies.40 The prevalence of preterm birth among patients with MDR-TB in our study (9.5%) was similar to the worldwide prevalence of preterm birth (11%), the prevalence of preterm birth among patients with drug-susceptible TB (8.0%), and the prevalence of preterm birth in a previous systematic review of MDR-TB among pregnant patients (12.9%).4 The prevalence of stillbirth in our study (1.9%) was also similar to that reported for patients with non-TB diseases, such as COVID-19 (1.4%),41 and among the general population (1.8%).43 However, the prevalence of low birth weight (3.9%) in our study was lower than the prevalence of low birth weight among patients with drug-susceptible TB (8.5%),44 in the general population (14.6%),46 and in a previous systematic review (23.6%). The reason for the difference in the prevalence of low birth weight between our systematic review and the previous systematic review could be the difference in the number of studies included in the systematic review. In the previous systematic review, only 2 studies were included, whereas in the present systematic review, 6 studies were included. The main reasons for pregnancy loss were miscarriage and therapeutic termination of pregnancy.Preterm birth, pregnancy loss, low birth weight, and stillbirth were the most common types of adverse pregnancy outcomes found in our systematic review. These findings are consistent with a previous systematic review among patients with drug-susceptible TB that reported that TB during pregnancy is associated with adverse pregnancy outcomes, such as low birth weight and stillbirth.This study has some limitations. Because only 10 studies with a total of less than 300 participants met our inclusion criteria, the metaregression model was underpowered to detect associations between study characteristics and successful treatment outcomes. There were no studies from the Southeast Asia region, India, Russia, or other countries with a high TB burden. In addition, the crude classification of study characteristics, such as stratifying analyses at the median value for the proportion of patients receiving a certain drug, may have resulted in misclassification of exposure for many patients treated with individualized regimens and may have reduced our chances of finding any associations with outcomes, even if present. Treatment success rates did not differ by TB medicines (except for linezolid), but the small sample size and the lack of individual patient data prevent us from drawing firm conclusions. Moreover, owing to the limited number of studies, we were not able to stratify adverse drug events based on medication types. Selection bias could also be a major limitation of our study because almost all of the studies included in our systematic review were conducted using retrospectively collected data. This review highlights the need for more standardized reporting, as well as evidence from well-designed randomized clinical trials, or a meta-analysis of pooled individual patient data from multiple observational studies among pregnant patients with MDR-TB.The findings of this meta-analysis and systematic review suggest that high treatment success rates and favorable pregnancy outcomes can be achieved when pregnant patients with MDR-TB are treated with effective regimens. Further research is needed to design shorter, more effective, and safer treatment regimens for pregnant patients with MDR-TB.


Lumbar spinal canal stenosis (LSS) and diffuse idiopathic skeletal hyperostosis (DISH) tend to develop in the elderly, resulting in an increased need for lumbar surgery. However, DISH may be a risk factor for poor clinical outcomes following lumbar decompression surgery, especially in patients with DISH extending to the lumbar segment (L-DISH). This study aimed to identify the prognostic factors of LSS with L-DISH and propose an optimal surgical management approach to improve clinical outcomes. Of 934 patients who underwent lumbar decompression surgery, 145 patients (15.5%) had L-DISH. In multivariate linear regression analysis of the JOA score improvement rate, the presence of vacuum phenomenon at affected segments (estimate: −15.14) and distance between the caudal end of L-DISH and decompressed/fused segments (estimate: 7.05) were independent prognostic factors. In logistic regression analysis of the surgical procedure with JOA improvement rate > 25% in L-DISH patients with both negative prognostic factors, the odds ratios of split laminotomy and short-segment fusion were 0.64 and 0.21, respectively, with conventional laminotomy as the reference. Therefore, to achieve better clinical outcomes in cases with decompression at the caudal end of L-DISH, decompression surgery without fusion sparing the osteoligamentous structures at midline should be considered as the standard surgery. Diffuse idiopathic skeletal hyperostosis (DISH) has an unknown etiology, but is a non-inflammatory condition with features of calcification and ossification of connective tissues, especially in the spinal ligaments . The preFurthermore, the presence of DISH has been considered to be a risk factor for lumbar posterior decompression surgery with and without fusion for LSS ,8,9,10. The aim of this study was to identify the predictors of poor surgical outcomes in LSS patients with L-DISH based on the Japanese Orthopaedic Association (JOA) score improvement rate at follow-up and to establish an approach for optimal surgical management to achieve better clinical outcomes with significant improvement.A total of 934 LSS patients underwent lumbar posterior decompression surgery without fusion or 1- or 2-level short-segment posterior lumbar interbody fusion (PLIF) between 2005 and 2019, and were followed up for >2 years. Patients who had undergone previous spinal surgery and those aged <50 years were excluded from the study. The Resnick criteria were used to diagnose DISH on lateral lumbar spine radiographs below T10 as follows: (1) ossification/calcification along the anterior aspect of at least 4 contiguous vertebrae, (2) a somewhat preserved intervertebral disc height, and (3) no sacroiliac joint erosion , and theThe JOA scoring system before and at follow-up, including those who underwent revision surgery within 2 years after surgery, was used to assess the clinical status. JOA score improvement rate was calculated using the following formula: (JOA score at follow-up—preoperative JOA score) × 100/(29—preoperative JOA score). Cases with a JOA score improvement rate of ≤25% were defined as a poor clinical outcome. The rate of revision surgery at the same segment and/or an adjacent segment was determined. Clinical data including age, gender, body mass index (BMI), and surgical procedures were obtained from medical charts. In L-DISH cases, preoperative plain radiography, computed tomography (CT), and magnetic resonance imaging (MRI) were used to examine if an intradiscal vacuum  or vertebral bone marrow edema ) was present at the decompression level . Lumbar We changed the surgical procedure of lumbar decompression from conventional laminotomy to lumbar spinous process-splitting (split) laminotomy in 2015 because of the advantages of a less invasive procedure for lumbar paraspinal muscles . In convp < 0.05 considered significant. Factors that were significant in univariate analysis and other factors from the literature were included in multivariate regression analysis. The estimate, odds ratio, and 95% confidence interval (CI) were calculated to identify independent predictors of JOA score improvement. To assess the inter- and intraobserver reliabilities, intraclass correlation coefficients (ICCs) were calculated. EZR  [Data are presented as the median [interquartile range]. Mann-Whitney U-test or chi-square test were used to compare the categorical variables, with , Japan) , which ip = 0.0050).A comparison of L-DISH patients with and without JOA improvement rate > 25% at the final follow-up is shown in Multivariate linear regression analysis of the JOA score improvement rate was performed to identify independent prognostic factors in L-DISH patients . The preTo clarify the surgical strategy for the affected segments with vacuum phenomenon at a lower segment or one distance from the caudal end of L-DISH, logistic regression analysis of the surgical procedure with JOA improvement rate > 25% was performed. Of the 145 patients, 44 patients (30.3%) were included in the analysis. Although no significant statistical differences were observed, the odds ratios of split laminotomy and short-segment fusion were 0.64 and 0.21, respectively, with conventional laminotomy as the reference .The high mechanical stress on segments free of ossification due to a longer lever arm at the distal end of L-DISH can induce disc degeneration or ligamentum flavum hypertrophy, which has been associated with a requirement for surgery for subsequent development of LSS ,9,10,11.The current study showed that the presence of vacuum phenomenon and distance of affected segments from L-DISH were independently and significantly associated with the JOA score improvement rate. In a previous study, CT findings revealed that a vacuum phenomenon was associated with the severity of disc degeneration and LSS, with an incidence rate of 5.6–9.2% at lumbar segments . In our Similarly to fusion surgery, decompression surgery without fusion has been reported to have a high revision rate . A higheThere are several limitations in this study. First, it had a retrospective and single-center design including a small number of patients. Second, the whole range of DISH was not assessed because plain radiographs of the thoracic spine above T10 were not assessed in all cases of this study. Third, further investigation is needed to define the optimal surgical procedure, including minimally invasive surgery such as endoscopic surgery  and longThe presence of a vacuum phenomenon and a short distance from the caudal end of L-DISH of the affected segment were negative preoperative prognostic factors in LSS patients with L-DISH. To achieve better clinical outcomes and prevention of revision surgery for these patients, short-segment fusion surgery is not recommended as standard surgery. It is advisable to perform decompression surgery without fusion, and a surgical procedure conserving spinous process and supra/interspinous ligaments at the midline should be selected, instead of lumbar spinous process-splitting laminotomy.


Statins, such as lovastatin, are lipid-lowering drugs (LLDs) that have been used to treat hypercholesterolaemia, defined as abnormally elevated cholesterol levels in the patient’s blood. Although statins are considered relatively safe and well tolerated, recipients may suffer from adverse effects, including post-statin myopathies. Many studies have shown that supplementation with various compounds may be beneficial for the prevention or treatment of side effects in patients undergoing statin therapy. In our study, we investigated whether L-carnitine administered to zebrafish larvae treated with lovastatin alleviates post-statin muscle damage. We found that exposure of zebrafish larvae to lovastatin caused skeletal muscle disruption observed as a reduction of birefringence, changes in muscle ultrastructure, and an increase in atrogin-1. Lovastatin also affected heart performance and swimming behaviour of larvae. Our data indicated that the muscle-protective effect of L-carnitine is partial. Some observed myotoxic effects, such as disruption of skeletal muscle and increase in atrogin-1 expression, heart contraction could be rescued by the addition of L-carnitine. Others, such as slowed heart rate and reduced locomotion, could not be mitigated by L-carnitine supplementation. Statins, e.g., lovastatin, simvastatin, and atorvastatin, belong to a family of lipid-lowering drugs (LLDs) that have been used for many years to combat one of the most common pathological conditions–hypercholesterolaemia, manifested as an elevated level of cholesterol in the patients’ blood. Statins, similar to other LLDs, allow the risk of cardiovascular diseases (CVDs) to be reduced. Statins are considered relatively safe and well-tolerated therapeutic drugs. However, their administration occasionally may induce adverse effects manifested in myotoxicity ,2,3. StaMolecular mechanisms underlying statin-induced myopathy involve down-regulation of PI3k/Akt signalling and up-regulation of FOXO transcription factor expression, which is associated with increased oxidative stress and inflammation in rat skeletal muscle . UpregulSupplementation of statin-exposed individuals with various compounds, including CoQ10, L-carnitine, and geranylgeraniol, has been suggested to be potentially beneficial in preventing or alleviating side effects in patients undergoing statin therapy ,10,11. FL-carnitine is an ammonium compound of amino acid origin, naturally occurring in animals. It is involved in energy metabolism via participation in the transport of long-chain fatty acids into mitochondria for their β-oxidation. L-carnitine plays a role in the removal of accumulated toxic fatty acyl-coenzyme A (acyl-CoA) metabolites and maintenance of the balance between free and acyl-CoA ,15. In aVarious studies have revealed that L-carnitine plays an important role in controlling energy metabolism and endurance capacity and can act as an antioxidant ,18,19. LWhat seems particularly interesting, in the context of preventing muscle damage through supplementation, is the fact that L-carnitine administration impedes muscle atrophy induced by prolonged hindlimb suspension in rats . L-carniAs mentioned above, L-carnitine appears to be a remedy worth considering for alleviating muscle damage of different origins, including post-statin muscle symptoms. However, especially in the case of post-statin muscle damage, to verify and thoroughly understand the mechanism of action of both statins, which trigger muscle damage and L-carnitine as a potential therapeutic, further studies, including research using model organisms such as zebrafish, need to be conducted. atrogin-1 expression, statins may lead to muscle fibre damage, developmental arrest, improper axis elongation, somite compression, changes in the muscle cytoskeleton and myofibril organisation, and pericardial oedema ) were randomly allocated to 50 mm Petri dishes filled with 5 mL per 10 individuals of freshly prepared experimental solutions. Animals were incubated at 28.0 °C for 24 h under standard photoperiod conditions. Larvae (120 hpf) were rinsed with embryo medium and collected for further experiments. The experimenter conducting particular experiments was not involved in collecting the larvae. The experimenter conducting particular experiments (morphology and birefringence assessments) were also blinded to the treatment groups.Muscle birefringence was analysed in 120 hpf zebrafish larvae (40–86 in each group). Animals were anaesthetized with 0.04% tricaine and placed on a depression glass microscope slide. While the polarising filters were crossed, the fish were rotated to find the angle that maximised birefringence. The microscope exposure was adjusted to see the light refracting through the trunk skeletal muscle of the vehicle control (VC) fish. All settings remained unchanged during the examinations of all control and investigated groups. The observations were performed, and images were acquired using the Leica DM5000 light microscope  with a pair of polarised lenses. ImageJ software was used to quantify the birefringence.For electron microscopic techniques, the 120 hpf zebrafish larvae were anaesthetized. Larva fixation, embedding, and sectioning were performed as described previously .Total RNA from investigated larvae was extracted using the Extracol reagent , according to the protocol provided by the manufacturer. RNA was quantified using the NanoDrop OneC Spectrophotometer . The cDNA was synthesised using a HighCapacity cDNA Reverse Transcription Kit .Real-time quantitative PCR (RT-qPCR) was performed using the CFX Connect Real-Time PCR Detection System  utilising the PowerUp SYBR Green Master Mix  with the gene-specific primers indicated below .rpl13a gene. The normalised number of atrogin-1 (fbxo32) molecules in the samples obtained from the 5 µm LOV treatment group was considered to be 1 arbitrary unit (A.U.).The software automatically determined the Ct values. Standard curves for each pair of primers were prepared by serial 5-fold dilutions of the template cDNA followed by the determination of reaction efficiencies. The number of atrogin-1 molecules was determined from the curve and then normalised to the The spontaneous displacement was examined according to a modified method from Xi et al. . Larvae The response of larvae to tactile stimulation was examined using a method modified by Granato et al. . Larvae The zebrafish larvae were anaesthetized and placed on a glass microscope slide. Next, 30-second movies were recorded with a focus on the heart area. The heartbeat analysis was conducted using a light microscope  under the 10× objective. The movies were analysed using Image J software with the time series analyser V3 plugin . The obtZebrafish larvae (5 in each group) were anaesthetized and placed on a glass slide. Videos of heart movements  were recorded under 4× magnification with a focus on the heart area . Then the movies were converted to AVI format, and the perimeter of the larva ventricle and atrium in systole and diastole were determined using Image J (3 times for each fish) . Three matrogin-1 expression level are given as means and standard deviations, and their significance was determined with Student’s t-test. Data concerning real-time quantitative PCR for Data regarding birefringence analysis and evaluation of heartbeat are given as means and standard deviations and were statistically analysed using the ANOVA test followed by the Games–Howell post-hoc test due to the lack of homogeneity of variance. Contraction of the heart muscle based on time-lapse images is given as means and standard deviations. Spontaneous displacement and touch-evoked response assay are expressed as a percentage of the larvae responding. All three tests were statistically analysed using the Kruskal–Wallis test followed by the Pairwise Mann–Whitney post-hoc test.p < 0.05 was used in all statistical analyses. At least three independent experiments were carried out. The data analysis for this paper was generated using the Real Statistics Resource Pack software (Release 6.8), copyright (2013–2020) Charles Zaiontz.A significance level of The 120 hpf zebrafish larva trunk muscle structure was observed and analysed using birefringence, a common non-invasive assay used to determine the degree of muscular disorganisation of zebrafish embryos during early development . The norThe morphology of most of the larvae treated with both lovastatin and L-carnitine showed no significant changes compared to those of all control groups. Of note, we did not observe any significant changes in analysed zebrafish larva morphology in transmitted light microscopy. Neither changes in the zebrafish trunk nor significant malformations in the pericardial region were observed .Previous studies on statins’ impact on zebrafish skeletal muscles involved analysis using light microscopy ,25,26. Tatrogin-1 expression. This phenomenon was observed in, inter alia, zebrafish embryos exposed to these compounds [atrogin-1 expression is also increased in 120 hpf zebrafish larvae treated with lovastatin. Our results obtained using real-time quantitative PCR (RT-qPCR) confirmed that the mRNA atrogin-1 expression level increased significantly after lovastatin administration in comparison to all control groups  had a significant alleviating effect, which was manifested by inhibiting the increase in the expression of atrogin-1, maintaining it at a level similar to those observed in the control groups  on skeletal muscle performance in zebrafish larvae, we carried out swimming behaviour tests  in larvae treated with these substances. Our analyses showed that zebrafish larvae treated with lovastatin exhibited significantly decreased spontaneous larval displacement in comparison to all control groups A,B. AdmiWe also examined the response of larvae to tactile stimuli. We observed that the response to tactile stimuli in larvae was significantly decreased, regardless of the method of stimulation (touching the tail or the head), in the lovastatin-treated group in comparison to all controls C. FurtheTo assess whether lovastatin exposure affects zebrafish larvae (120 hpf) heartbeat performance, we decided to perform analyses that included evaluations of heart rate. Our studies revealed that lovastatin significantly reduces heartbeat rates in comparison to all control groups . We also evaluated the heartbeat parameter in relation to L-carnitine supplementation as a substance with the potential to mitigate the negative effects of lovastatin. Our study did not show that L-carnitine had a protective effect on the heartbeat in lovastatin-treated individuals. Administration of L-carnitine at the two investigated concentrations (100 and 200 μM) to lovastatin-administered zebrafish did not help restore the heartbeat rate observed in the control groups. In both tested groups (LOV+LC100 and LOV+LC200), the reduction in heartbeat was significant and similar to that observed in the LOV group .Further evaluation of the effect of lovastatin exposure on heart performance included analysis of its contraction. Separate analyses of ventricular and atrial contractions were performed. Our studies revealed that lovastatin significantly reduced both atrial and ventricular contractions in comparison to all control groups  . We also evaluated ventricular and atrial contractions in relation to L-carnitine supplementation as a substance with the potential to mitigate the negative effects of lovastatin. In this case, our study showed that L-carnitine exposure exhibits a protective effect in lovastatin-treated individuals. Administration of L-carnitine at both investigated concentrations (100 and 200 μM) to lovastatin-exposed larvae positively influenced ventricular contraction, bringing it back to the level observed in the control groups. In contrast, atrial contraction was restored to a level similar to that observed in the control groups in larvae treated with a higher L-carnitine concentration  treated overnight with either lovastatin or simvastatin exhibited abnormalities that comprise developmental arrest, improper axis elongation, and compressed somites .Serious morphological malformations were also reported as a result of high doses (0.375 to 1 μM) of simvastatin administration in embryos at the age of 24 or 30 hpf  . FurtherOther research groups have determined that the exposure of zebrafish larvae (80 hpf) only to a relatively high  dose of simvastatin significantly increased the percentage of individuals with abnormal morphology , as well as pericardial oedema . AdminisIn our study, we observed lovastatin-associated skeletal muscle abnormalities, but not to the same extent as researchers who conducted their examination using zebrafish at earlier embryonical developmental stages ,25,26,31To better understand the nature of lovastatin-induced muscle damage, we decided to perform an examination using the electron microscope. This analysis showed that lovastatin exposure leads to abnormalities of the contractile apparatus . L-carniatrogin-1, considered a useful marker of muscle atrophy. Our RT-qPCR studies revealed that lovastatin enhances the expression of atrogin-1 in zebrafish larvae (atrogin-1 mRNA level is increased by lovastatin in a time- and concentration-dependent manner. At the highest used lovastatin dose (10 μM), the increase in atrogin-1 mRNA level was 6-fold when compared to the control group. The increase in transcript level was mirrored in protein level growth. The authors also observed a two-fold increase in atrogin-1 expression in the statin-treated patient’s muscle biopsy compared to non-treated individuals. Their investigation of atrogin-1 expression in zebrafish embryos treated with lovastatin (0.5 μM) for 12 h also revealed its increase.We also assessed the muscle damage process at the molecular level by examining via RT-qPCR the expression of h larvae . This isatrogin-1 expression without the possibility to distinguish any differences in mRNA expression with consideration to specific tissues, e.g., skeletal or cardiac muscle.The data presented in our manuscript suggest that atrogin-1 may be an appropriate marker for lovastatin-mediated muscle atrophy also in the zebrafish larvae. However, since for each sample preparation, we used 30 whole zebrafish larvae, we could only assess the global changes in atrogin-1 expression compared to levels that occurred in individuals with symptoms of muscular atrophy induced by prolonged hindlimb suspension [As with other analyses carried out in this work, the second part of our RT-qPCR analysis involved assessing the potentially protective effects of L-carnitine depending on the concentration used. The results show that administration of L-carnitine at a higher dose (200 μM) to lovastatin-exposed larvae inhibits the increase in the expression of atrogin-1 level and maintains it at a level similar to those in the control groups . This isspension .atrogin-1 level was differentially affected by atorvastatin exposure, i.e., it was significantly increased at concentrations of 0.081 and 1.8 μM, but significantly decreased at 0.9 μM in comparison to the control group. Atrogin-1 levels showed the same trend following CoQ10 administration, indicating no rescue effect [atrogin-1 levels obtained with doses of 0.9 and 1.8 μM could be associated with the dynamic gene expression during larval development [Interesting results were obtained by researchers carrying out studies on zebrafish larvae exposed to atorvastatin and/or CoQ10 . The atre effect . The autelopment . To better and more broadly assess the condition of zebrafish skeletal muscles, swimming behaviour tests are often performed. Negative effects of various statins on zebrafish swimming behaviour have been found in both embryos and larvae. For example, a lower dose (0.3 nM) of simvastatin significantly impaired the swimming capacity of 48 hpf zebrafish embryos, while cholesterol was able to compensate for the effects of simvastatin . In turnAs mentioned earlier, statins disrupt the function and structure not only of skeletal muscles but also of the heart. One of the potential symptoms of heart failure is pericardial oedema ,33. AlthPericardial oedema was observed in younger embryos (48 hpf) in response to a low dose (0.3 or 0.6 nM) of simvastatin treatment . FurtherThe data mentioned above are consistent with our results, which showed that lovastatin significantly lowers heart rate  and alsoThese discrepancies may be due to a too low dose of L-carnitine used in our experiments. Supporting this supposition is the fact that when examining cardiac contraction, we noted that the higher dose (200 μM) had a significant protective effect on both atrial and ventricular contraction, whereas the lower dose (100 μM) was effective only for ventricular contraction .atrogin-1 expression in comparison to all control groups. The research established that all observed myotoxic effects could be rescued by the addition of L-carnitine. We also found that lovastatin exposure has a significant impact on larval heart performance tested for the heartbeat frequency and changes in ventricular and atrial contractions. This impact was manifested by a significant decrease in all examined parameters. Intriguingly, our results indicate that, in this case, L-carnitine exhibits only a partially protective effect by rescuing ventricular and atrial contractions without restoring the heartbeat to a normal level. We also discovered that lovastatin exposure changes zebrafish swimming behaviour by reducing the response to tactile stimuli in larvae and that this effect could not be rescued by the addition of L-carnitine.Our study showed that the myotoxic effects of lovastatin on 120 hpf zebrafish larvae manifested in a significant reduction of birefringence, changes in muscle ultrastructure, and a significant increase in Although our study contributes to a better understanding of the mechanisms of action of statins showing the myo- and cardiotoxic effects of lovastatin on zebrafish larvae and providing interesting clues to the protective effects of L-carnitine, further studies, focused on, inter alia, mitochondria condition and oxidative stress, as well as on lovastatin-induced cardiotoxicity, are needed to deliver more details on the molecular basis of our observations.