COVID-19 restrictive containment was responsible for major psychological distress and alteration of quality of life (QoL) in the general population. Their impact in a group of patients having cerebral small vessel disease (SVD) and at high risk of stroke and disability was unknown.We aimed to determine the potential psychological impact of strict containment during the COVID-19 pandemic in a sample of CADASIL patients, a rare SVD caused by NOTCH3 gene mutations.Interviews of 135 CADASIL patients were obtained just after the end of the strict containment in France. Depression, QoL and negative subjective experience of the containment were analysed, as well as predictors of posttraumatic and stressor-related manifestations, defined as an Impact Event Scale-Revised score ≥ 24, using multivariable logistic analysis.Only 9% of patients showed a depressive episode. A similar proportion had significant posttraumatic and stressor-related disorder manifestations independently associated only with socio-environment factors, rather than clinical ones: living alone outside a couple (OR 7.86 (1.87–38.32), unemployment (OR 4.73 (1.17–18.70)) and the presence of 2 or more children at home (OR 6.34 (1.35–38.34).Psychological impact of the containment was limited in CADASIL patients and did not appear related to the disease status. About 9% of patients presented with significant posttraumatic and stressor-related disorder manifestations which were predicted by living alone, unemployment, or exhaustion related to parental burden. In France, at the heart of the COVID-19 epidemic in 2020 and well before the emergence of vaccination, major psychological distress and significant impairment of quality of life occurred in the general population at time of the containment , 27. ThePredicting the negative impact of containment on the quality of life and psychological health was then crucial for developing preventive strategies at individual or familial level or specific policies at population level , 42, 47.At the end of the strict containment in France, we chose to investigate patients with a genetically-confirmed diagnosis of Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL). CADASIL is caused by mutations of the NOTCH3 gene that stereotypically lead to vascular accumulation of the derived protein products around smooth muscle cells and pericytes in the wall of cerebral microvessels , 22. TheAt the beginning of the COVID-19 pandemic, CADASIL patients were informed that they were particularly exposed to an increased risk of stroke , 43, 46 The present study aimed at estimating the importance of distress in CADASIL patients during this exceptional event and to delineate potential predictors.www.cervco.fr); (4) having at least two previous follow-up visits in the centre, (5) lack of sensory or severe cognitive difficulties  that could compromise the understanding of questions, (6) French language skills allowing easy understanding of questions asked by phone, (7) informed consent already obtained for collecting clinical and imaging data during follow-up. Patients already known having dementia (defined by DSM-IV-TR criteria  nor to the level of contamination in the patient’s environment  . A fraction of 15 to 20% of the sample had a level of 1, a similar fraction had a level 2, 3 and 4. Less than 5% did not experience any negative feeling. The level of this negative subjective experience was not related to the number of stressors presumably related to the negative impact of containment in the general population [(spearman rank (n = 12) were suffering from a major depressive episode. Two among the latter, who did not previously experience depression, were using an antidepressant treatment. Only three already experienced depression but were not treated. Of the 21 patients who were depressed at the last visit before the interview, only 6 continued to use an antidepressant, 18 were no longer affected during the containment period.At the time of the survey, only 9% of patients (n = 28) were currently using psychoactive drugs. When they were treated by an antidepressant drug, more than 90% of patients were using a selective serotonin reuptake inhibitor (SSRIs) . Only 3% of individuals were daily using anxiolytics (benzodiazepine drugs) and only one patient was treated by an antipsychotic drug  = 0.08, p = 0.02), a small but significant increase of the mRS score  and of the MADRS score  was detected at time of the survey. Conversely, the variation between pre and post-containment of the Euroquol global visual score and of the Euroquol quantitative score evaluation of anxiety/depression dimension did not vary significantly in the whole sample .Although depression was less present in the whole sample after the containment then before . Only 13 patients, had an IES-R score larger than or equal to 24. Among them, 7 had a score equal or higher than 33. These two groups were compared to patients with IES-R less than 24 (Table p < 0.03), (2) the status of being single and not in couple  and 3) the fact to have more than one child  at home during the containment  included 4 factors, 3 of which were predictors of significant post-stress manifestations: (1) the status of unemployment (OR = 4.73, The results of this study showed that the strict containment ordained in France over 8 weeks in 2020 to reduce the COVID 19 contamination had, globally, a limited impact on mental health in a sample of CADASIL patients. Just at end of the containment, only 9% of individuals were found depressed whereas this frequency was about 16% at their last follow-up visit. More specifically, 18 patients who were previously depressed were no longer affected at time of the containment. This low prevalence contrasts with the 16.5% frequency of depression estimated in a survey obtained in the general population at the initial outbreak among 1738 respondents from 190 Chinese cities . It was Exposure to an exceptional event as the “lockdown” could also have precipitated transient or more enduring psychological manifestations related to posttraumatic stress or stressor-related disorder independently from depression . The meaCADASIL patients with posttraumatic or stressor-related manifestations were found more frequently to be women, living alone, unemployed, and having more neurological disability in univariate analysis. However, only 3 socio-environmental factors predicted independently the occurrence of posttraumatic or stressor-related manifestations. All these factors had been already recognized as potentially capable to precipitate the occurrence of stress manifestations during the COVID 19 pandemic in the general population , 41, 47.We are fully aware of different weaknesses of the present study. The selection of our patients was necessarily biased because we chose to investigate only individuals from a cohort of volunteers, who already accepted to be followed and for whom information could be easily collected. Thus, this sample could not represent the whole population of CADASIL patients. Moreover, the study population was relatively small in size which prevented the identification of other predictors of post-containment manifestations. We also did not validate externally the present results in an independent population. Thus, it is most likely that the present findings might not be transposed to a different CADASIL cohort in a distinct social, cultural or political context. For instance, the lack of predictors related to the clinical or psychological status observed in the present study might be specifically related to the situation of individuals who were diagnosed in France, who could benefit from a 100% coverage of all their health care costs as well as a full wage replacement throughout the entire period of containment. The present study also had a number of strengths. All investigations were carried out in a homogeneous population corresponding to patients at the highest risk of decompensation who already had a confirmed genetic diagnosis of their disease and without major disability. This study was also based on clinical tools and scores already validated. Detailed clinical information were available for each individual. Clinical and psychological information were updated and collected through direct intervention of professionals with a long expertise in the management of CADASIL. The results were also coherent. They showed without any ambiguity the importance of socio-environmental factors that could worsen the psychological status of CADASIL patients during the lockdown period.In conclusion, we believe that the results of this study support that isolation of CADASIL patients, unemployment and the number of children at home should be considered to evaluate the risk of psychological distress during exceptional situations leading to social breakdown as the containment.


Acute blast or gunshot wounds have a negative effect on the patients’ health related quality of life (HRQoL). No validated instrument exists to assess the HRQoL of patients with such wounds. Therefore, we aimed to test and validate a subscale of an existing HRQoL instrument among patients with acute blast or gunshot wounds.body subscale of the Wound QoL instrument using the World Health Organisation 20 question self-reporting questionnaire as gold standard.We used data from a randomized controlled trial comparing negative pressure wound therapy with standard treatment of civilian adults with acute extremity blast or gunshot wounds. We evaluated the reliability  and validity of the A total of 152 participants were included in the study. The participants were predominantly (93.4%) male, and median age was 29.0 years (IQR 21.0–34.0). The internal consistency was acceptable while a test-retest analysis indicated instability in the Wound QoL instrument. The content validity of the instrument was considered satisfactory; however, the criterion validity was found to be insufficient.Our results indicate that Wound QoL is a promising instrument for the assessment of wound specific HRQoL among patients with acute blast or gunshot wounds. Further testing and validation is needed. The global number of direct fatalities caused by armed conflict and terrorism is increasing, reaching an estimated 129 700 in 2017 , 4. The HRQoL in patients with wounds can be measured either with generic tools or with wound specific tools. One of the first wound specific HRQoL tools was the 47-item Cardiff Wound Impact Schedule (CWIS) , 9. TherTo optimize and individualize the care it is important not only to measure treatment efficacy and safety but to also focus on the patients´ experience of their condition and treatment . There iWe used data from a randomized controlled trial comparing negative pressure wound therapy (NPWT) with standard treatment of adults (≥18 years) with acute (≤72 h) extremity blast or gunshot wounds  . ParticiThe Wound QoL instrument is developed and validated to assess disease-specific HRQoL of patients with hard-to-heal wounds . The insThe World Health Organisation 20 question self-reporting questionnaire (SRQ-20) was originally developed as a tool to screen for mental disorder , and hasWe evaluated the reliability of the “body” subscale of the Wound QoL instrument. To test reliability in regard to internal consistency we estimated the correlation between the four subscale items at baseline by using Cronbach´s alpha coefficient. A Cronbach´s alpha coefficient of 0.7 or higher was considered acceptable . We alsoWe assessed validity according to content validity and criterion validity. Content validity refers to the extent to which a measure covers relevant areas of the condition or disease . CriteriCeiling effect occurs when the scores of an instrument reach the maximum possible score. Floor effect occurs when the scores are equal to the minimum possible score. If ceiling or floor effect occurs in 15 percent or more of the questionnaires, it is considered hard to detect improvement or decline in the rated health status .® Statistics .We analysed data using SPSSThis study was part of a larger clinical trial , which rA total of 152 participants were included in the study. The participants were predominantly (93.4%) male, and median age was 29.0 years (IQR 21.0–34.0). Some 99/152 (65.1%) participants were smokers, and 13/152 (8.6%) participants had prior diseases, among which one had diabetes mellitus. Wounds in 94/152 (61.8%) participants were caused by gunshots, and in 58/152 (38.2%) participants by bomb blasts. The Cronbach´s alpha coefficient was 0.7. Test-retest analysis was performed on questionnaires from 90 participants with an ICC of 0.47.Content validity was discussed within the expert group and the four items constituting the “body” subscale were found to be valid and relevant for the studied patient group. The criterion validity assessments included all 152 participants. Spearman´s rho was 0.20.We found no signs of ceiling effect as no participant received the highest score. Some 19/152 (12.5%) participants had minimum scores, indicating acceptable levels regarding floor effect.Our results indicate that the “body” subscale of the Wound QoL instrument could represent a reliable and valid tool for the assessment of physical aspects of wound specific HRQoL among patients with acute blast or gunshot wounds. The internal consistency of the subscale items was acceptable; however, the test-retest analysis indicated instability in the Wound QoL instrument. This instability might be partially explained by a rapid clinical improvement among patients with an acute traumatic wound, compared to a slower change of HRQoL among patients with hard-to-heal wounds. One way to achieve a more reliable test-retest analysis could be to distribute the second questionnaire (re-test) closer in time to the first one.We demonstrate that the Wound QoL instrument may be able to detect improvement and deterioration in HRQoL since we did not find any ceiling or floor effect. The content validity of the instrument was considered satisfactory by the expert group. However, the criterion validity was found to be insufficient, indicating that the use of SRQ-20 as gold standard for quality of life measures was suboptimal. When designing the study, we opportunistically decided to use SRQ-20 for our analyses as one of the study sites had already implemented the instrument as a standard screening tool. Further, the instrument is well-established and used worldwide in emergency settings in conflict areas. To assess its use in the present analyses was therefore deemed to be of value, in particular considering the lack of instruments aimed at measuring HRQoL in patients with acute wounds.The impact of acute and traumatic wounds on patients´ HRQoL is scarcely described in the literature. One small study published in 2000 found that the patients with acute wounds rated their overall HRQoL higher compared to the patients with hard-to-heal wounds . The autSome limitations should be considered. First, the sample size is rather small. However, to prospectively include study participants with acute blast or gunshot wounds is a challenge. Particularly at low-resource or conflict settings. Second, only the “body” subscale of the Wound QoL instrument was used. However, this was the only of the three subscales that was considered relevant. The main limitation is the use of SRQ-20 as gold standard. The SRQ-20 instrument is mainly used as a screening tool for mental disorder. Still, we anticipated that the instrument items measuring somatic symptoms would prove to be transferrable to the physical aspects of HRQoL. The low criterion validity indicates that this might not be the case. Bearing the above mentioned limitations in mind, we still believe that the present study adds important knowledge on the utility and limitations of the SRQ-20 instrument, considering its widespread use.The “body” subscale of the Wound QoL is a promising instrument for the assessment of wound specific HRQoL among patients with acute blast or gunshot wounds. Future research in patients with acute traumatic wounds could be aimed at how to differentiate between the HRQoL effect generated by the psychological trauma and the effect generated by the actual wound.


Although speech declines rapidly in some individuals with amyotrophic lateral sclerosis (ALS), longitudinal changes in speech have rarely been characterized. The study objectives were to model the rate of decline in speaking rate and speech intelligibility as a function of disease onset site, sex, and age at onset in 166 individuals with ALS; and estimate time to speech loss from symptom onset. We also examined the association between clinical (speaking rate/intelligibility) measures and patient-reported measures of ALS progression (ALSFRS-R). Speech measures declined faster in the bulbar-onset group than in the spinal-onset group. The rate of decline was not significantly affected by sex and age. Functional speech was still maintained at 60 months since disease onset for most patients with spinal onset. However, the time to speech loss was 23 months based on speaking rate < 120 (w/m) and 32 months based on speech intelligibility < 85% in individuals with ALS-bulbar onset. Speech measures were more responsive to functional decline than were the patient-reported measures. The findings of this study will inform future work directed toward improving speech prognosis in ALS, which is critical for determining the appropriate timing of interventions, providing appropriate counseling for patients, and evaluating functional changes during clinical trials. The onset and progression of dysarthria, the speech disorder associated with ALS, is a major concern of patients who invariably want to know “when” and “how fast” their loss of speech will occur. Despite the critical importance of accurate speech prognosis to clinical care, the longitudinal decline of speech has rarely been characterized, leading clinicians to speculate based on their selective and subjective experiences. Improving predictive models of speech decline is not only needed to better advise patients regarding the expected course of their disease, but also critical for optimizing ALS clinical trials and treatment strategies for maintaining communication abilities throughout the disease.Most individuals with amyotrophic lateral sclerosis (ALS) progressively lose the ability to speak and, therefore, eventually require assistive devices to fulfill communication needs9, the focus on speech decline has been limited. Studies of speech are needed because the rate of speech decline may be decoupled from that occurring in other parts of the motor system5 and the disease progression and expression are known to be very heterogeneous13. The few existing studies of longitudinal speech decline in ALS suggest that speech remains functional for an average of 18 months from the first bulbar symptom14. For individuals who experience their first symptom in bulbar muscle , speech tends to decline faster than for those with spinal-onset ALS14 and can decline as fast as 20% a year15. While these estimates are useful, they are derived from only a few studies, some of which had design issues that limit interpretation, including a relatively small number of participants. For instance, the estimates of speech decline in Makkonen et al.14 were based on only 9 participants  who completed their follow-up session. In addition, the model of speech intelligibility decline provided by Rong et al.15 did not account for site of onset and was calculated in months from ALS diagnosis, which may be problematic as participants could be at different stages of their disease at the time of diagnosis. Ideally, predictive models of ALS speech decline will account for the influence of potential risk factors such as site of disease onset, age at onset, and sex on group and individual trends. Additional research is also needed to identify the most responsive markers of speech decline.Although the course of overall functional decline due to ALS has been modeled extensively using clinical scales such as the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R)16. During the course of speech decline, it has been reported that (1) changes in speaking rate precedes the onset of intelligibility decline in individuals with ALS, and (2) intelligibility declines in a faster rate when speaking rate passes the threshed of 120 w/m17. Further, speaking rate is significantly associated with clinical measures of overall ALS and bulbar disease severity, and has necessary psychometric properties ) to detect bulbar signs prior to the onset of overt bulbar symptoms 18. The aims of the study were to (1) identify the status of speaking rate and speech intelligibility in ALS subgroups at baseline relative to normative data accounting for sex and age; (2) determine the rate of decline in functional speech measures, while controlling for ALS site of onset, age at onset, and sex; (3) estimate the time to speech loss in ALS subgroups; (4) compare the slope of change in speech measures to the slope of change in self-reported ratings ; and (5) determine if the decline in speech measures are associated with the decline in the ALSFRS-R total score and bulbar subscore. The comparative analysis between speech measures and ALSFRS-R scores is important because ALSFRS-R scores are commonly used in routine clinical evaluations, clinical trials, and patient-oriented research to estimate the overall progression of ALS. Results from this work may help clinicians improve estimates of the rate of speech decline and, as such, optimize patient care and the design of individualized treatment plans.In the current study, we used measures of speaking rate and speech intelligibility as indices of speech function because of their widespread use in clinical evaluation of bulbar function, disease severity, and motor speech impairment in ALS. Bulbar involvement is often characterized by slowed speaking rate and reduced speech intelligibilityM = 58 years, SD = 10) were included in this study. All participants in the ALS group were diagnosed with ALS by a neurologist using the revised El Escorial criteria19 and had no history of other neurological disorders. The ALS group was subdivided by site of symptom onset. Thirty participants  exhibited bulbar onset, while 136 participants  exhibited spinal onset. Unequal sample sizes in the ALS-Spinal and ALS-bulbar groups are expectable given the higher prevalence of ALS-spinal compared to ALS-bulbar onset. It’s been reported that the prevalence of ALS patients being characterized as spinal onset predominates that of bulbar onset by almost 58–82%20. In addition, 167 healthy controls (HC)  aged from 29 to 77 years old  were included as controls. Control group participants had no history of any known neurological, cognitive, speech, or swallowing impairment. Healthy controls volunteered to participate in the study and were recruited through flyers and Ads.One hundred sixty-six individuals with ALS  aged from 33 to 86 years old , University of Nebraska, and University of Dallas. The study sites located in Canada included the Sunnybrook Research Institute in Toronto and the sites involved in the Canadian ALS Neuroimaging Consortium (CALSNIC)22 by each participant to obtain more accurate estimations of the rate of decline in speech measures. In fact, the change score is often used in clinical trial settings. For example, the combined assessment of function and survival (CAFS) score is a well-accepted method of analyzing clinical trial data that looks at the change between the first and last measurements23. Therefore, all the 166 participants with ALS had both the baseline and follow-up data and, there were no dropouts due to death or loss to follow-up. The time interval between the two sessions ranged from 1 to 37 months .Participants were seen across multiple sessions during which they recorded a variety of speech and non-speech tasks. There was a large variation across participants in terms of the number of data collection sessions . In this heterogeneous dataset, a substantial portion of the participants had only two data points. Because creating linear models that have many data points for some individuals and only two data points for a large portion of individuals introduces model complexities and leads to overfitting, we analyzed changes between the first and last recordings of the Sentence Intelligibility Test (SIT)22. The number of words in each sentence ranged from five to 15. Participants were asked to read the sentences using their habitual speaking rate and loudness. Audio recordings of the participants were collected using the procedure reported in previous studies15. Subsequently, a trained research assistant who was unfamiliar with the SIT sentences and blind to the participants' diagnoses, listened to the audio recordings and transcribed the sentences produced by the participants. Speaking rate was calculated for each sentence as the number of words per minute (w/m) averaged across the 11 sentences. A percent intelligibility score for each sentence was calculated as the percent of intelligible (correctly transcribed) words out of the total number of words contained in the sentence. The percent intelligibility scores of all 11 sentences were averaged to derive the total percent intelligibility score. The inter and intrarater reliability of SIT transcriptions were reported in a recent study by our lab, which used a subset of data used in this study to create an empirical classification system for speech severity in patients with dysarthria secondary to ALS. Overall, the intraclass correlation (ICC) for intrarater transcription reliability was reported as 0.91 (p < 0.001) and the ICC for interrater transcription reliability ranged from 0.92 to 0.96 (p < 0.001)24, demonstrating excellent reliability in SIT transcription.Measures of speaking rate and speech intelligibility were extracted from recordings of each participant’s reading of 11 randomly generated sentences from the SIT25. It is commonly used in clinics to assess ALS disease progression and as an endpoint in ALS clinical trials and survival outcomes28. A measure of patient-reported bulbar function was derived from the ALSFRS-R bulbar subscore, which includes three items: speech, swallowing, and saliva management, with possible scores ranging from 0 (maximum disability) to 12 . Please refer to Table The ALSFRS-R is a well-established and reliable measure of global motor  disability in patients with ALSANOVA test was used to examine whether there were any differences between the groups  in terms of the age of participants. In addition, the proportion of male and female participants in each group was examined using chi-squared tests.Aim 1 examined the ALS subgroups’ speaking rate and speech intelligibility at baseline relative to normative data accounting for sex and age. This aim facilitates interpretation of the longitudinal results by showing, where the individuals with ALS stand in terms of speech functions at baseline relative to normative data and how their speech change at various stages of the disease. Separate multiple regression analyses were used to examine the effect of group, sex, and age on speaking rate and speech intelligibility. In the regression models, the variable group had three levels , and the variables sex, and age were binary and defined in two levels of analysis as follows: sex [males vs. females]; age at onset [younger than 60 years vs. older than 61 years]. The variable “age” was dichotomized because the cut-off point of 60 years yielded relatively equal sample sizes across the groups  × time as fixed effects and “subject” as the random effect. In the next step, fixed effect variables that were not statistically significant were removed from the LMEs models using a backward selection approach. Subsequently, a simplified LMEs model was produced for the effect of site of onset on the rate of decline in speaking rate and intelligibility irrespective of sex and age (non-significant variables). In LMEs models, the time scale was based on months relative to baseline.Aim 2 determined the rate of decline of functional speech measures while accounting for clinical and demographic factors. To address this aim, the lme function from the nlme packages in R statistical software (i386 3.6.1), was used to fit linear mixed effects (LMEs) models on longitudinal speaking rate and intelligibility data. LMEs models were used to mitigate the effect of unequal time-interval between the baseline and follow-up sessions, as well as missing and unbalanced data, on the reliability of slope of change in the outcome measuresBecause the baseline and follow-up sessions occurred at different time points relevant to the onset of ALS symptoms across all participants, the time (in months) of baseline and follow-up sessions were subtracted from the baseline time (in month) to align the baseline data for all subjects to 0 month from symptom onset. For example, if the baseline and follow-up sessions occurred at 5 and 12 months after symptom onset, the time points were shifted to 0 and 7 months, respectively. This technique allowed us to estimate the net rate of speech decline per month in each ALS subgroup . In the LMEs models, the variables of onset site, sex, and age were dichotomous and defined in two levels of analysis as follows: site of onset [spinal vs. bulbar]; sex [males vs. females]; age at onset [younger than 60 years at symptom onset  vs. older than 61 years at symptom onset ].31. Kaplan–Meier test was used to estimate the likelihood of maintaining speech functions based on the time to the occurrence of speaking rate below 120 (w/m), and intelligibility below 85%. These thresholds were selected based on the literature suggesting that speaking rate is a significant predictor of the intelligibility decline17 such that the slowness of speaking rate to approximately 120 words per minute (w/m), marks the initiation of accelerated decline in speech intelligibility (below 85%). Kaplan–Meier tests were fitted on the follow-up data (not the baseline) to estimate the fraction of individuals with ALS who maintain speech function until the follow-up session. Because all participants used in the study completed their follow-up session, there were no missing values due to mortality in our Kaplan–Meier analyses. In survival analysis models, the time scale was based on months since symptom onset which has been shown to be the best timescale in longitudinal studies of ALS, rendering models with increased precision and the lowest AIC32.Aim 3 sought to estimate the time to speech loss. Speech loss was defined as either speaking rate of below 120 (w/m) or speech intelligibility below 85%Aim 4 examined the difference in slope of change between speech measures and self-reported measures of ALSFRS-R . To address this goal, separate pairwise t-tests were used for individuals in ALS-bulbar and ALS-spinal subgroups to compare the slope of change in speaking rate and intelligibility to the slope of change in the ALSFRS-R total score and ALSFRS-R bulbar subscore. The slope of change in speech and ALSFRS-R measures were calculated by subtracting the measures obtained at follow-up from measures obtained at baseline divided by the time interval. The p-value was adjusted  and self-report ratings  across disease progression. The associations between the slope of change in functional speech measures and the slope of change in the ALSFRS-R total and bulbar subscores were assessed using the Pearson correlation tests. Bonferroni correction was performed to adjust the p-value in multiple tests.The variable age demonstrated normal distribution in all groups as displayed in Fig. Multiple regression analyses revealed significant effect of group on both speaking rate and intelligibility Table . NeitherResults of LMEs models for speaking rate and intelligibility demonstrated a significant effect of site of onset and a significant time × site of onset interaction Table . ResultsKaplan–Meier tests demonstrated significantly reduced likelihood of maintaining speech functions at 12, 24, 36, 48, and 60 months since symptom onset for individuals with bulbar-onset compared to those with spinal onset Fig. . While tIndividuals in the ALS-spinal group were more resilient to intelligibility decline than speaking rate decline over the 60-month follow-up. About 80% of individuals in the ALS-spinal group maintained intelligibility greater than 85% during the 60-month follow-up period. However, the median time to exhibiting intelligibility lower than 85% was 32 months from symptom onset for the ALS-bulbar group.In individuals with bulbar-onset ALS, the slope of change in both speaking rate and speech intelligibility were observed to be significantly faster than the slope of change in their ALSFRS total score and bulbar subscore. In individuals with spinal-onset ALS, however, the only significant difference in slope of change was between speaking rate and ALSFRS-R bulbar subscore, such that speaking rate declined faster than the ALSFRS-bulbar score in that group Table .Table 5Mr = 0.50  in the bulbar-onset ALS group compared to the spinal-onset ALS group at baseline. In addition, the rates of decline in speaking rate and intelligibility were faster in the bulbar-onset ALS group  than in the spinal-onset ALS group . Although the rate of decline varied significantly across individuals, the median time to speech loss defined as speaking rate slower than 120 (w/m) or speech intelligibility below 85% was relatively short for those with bulbar-onset ALS. While the median time to exhibit a speaking rate slower than 120 (w/m) and ineligibility below 85% was respectively 23 and 32 for the bulbar-onset ALS group, functional speech was still maintained for the majority of individuals in the spinal-onset group over 60 months since symptom onset. In fact, at 60 months (5 years) after symptom onset, 60% of participants with spinal-onset ALS exhibited a speaking rate greater than 120 (w/m) and 80% of them exhibited speech intelligibility greater than 85%. Finally, clinical speech measures  were more responsive to functional decline than were the patient-reported measures . The findings of this study have important implications for future work directed toward improving speech prognosis in ALS, which is critical for determining the appropriate timing of interventions, providing appropriate counseling for patients, and evaluating functional changes during clinical trials.25 as a prognostic index to assess global function in patients with ALS and its rate of decline33. Consistent with these self-reported measures, our models demonstrated significantly faster decline of speaking rate and intelligibility in individuals with bulbar-onset ALS. Additionally, our findings are consistent with previous findings of significant differences in speaking rate and/or intelligibility decline for those with bulbar vs. spinal-onset ALS34.Literature on the effect of onset site on ALS progression has consistently reported that patients with bulbar-onset ALS demonstrate deterioration of functional measures  at a markedly increased rate compared to individuals with spinal-onset ALS. The majority of these studies used the ALSFRS-R36. Speaking rate has been found to be a significant predictor of intelligibility decline, with the accelerated decline in speech intelligibility (below 85%) not occurring until speaking rate has slowed to approximately 120 w/m17.Our models also confirm prior work suggesting that speaking rate declines with a steeper slope than speech intelligibility over the course of the disease regardless of site of onset38, findings of this study suggested that rates of speech decline are driven primarily by site of symptom onset and are not influenced by sex or age. Our findings converge with Makkonen et al.14, who reported that among onset site, sex, and age, only the onset site significantly predicted the rate of decline in speech measures of individuals with ALS.Although several studies on models of ALS disease progression have attributed a faster rate of functional decline in older patients and females than in younger patients and males10 and exemplify the need for improved patient stratification. Therefore, objective criteria such as imaging-based biomarkers39, brain/plasma proteomics40, or assessment of bulbar motor involvement13 are necessary.The rate of speech decline for some individuals in both the spinal and bulbar groups diverged significantly from that of the overall group trend . Although the divergence only occurred in a small number of cases in each group, these findings provide further evidence in support of motor phenotype heterogeneity in ALSResults of this work suggest that speech measures can be used to uncover covert patient subgroups within the ALS-spinal and ALS-bulbar groups. Stratifying individuals with ALS into clinically relevant subgroups contributes to a better understanding of disease mechanisms and prognosis. In addition, subgroup analyses may enhance the quality of care for ALS patients by informing decision making, optimizing clinical trial planning/interpretation, and contributing to the development of effective therapeutic options.42. An important finding of this study was that the median time to speech loss based on speaking rate slower than 120 (w/m), was approximately 23 months for individuals with bulbar-onset ALS. In contrast, 60% of individuals with spinal-onset ALS were observed to maintain functional speech at 60 months since symptom onset. If we use speech intelligibility below 85% to index speech loss, the median time to speech loss was 32 months since symptom onset for individuals with bulbar-onset ALS. At 60 months since symptom onset, 80% of participants with spinal-onset ALS maintained speech intelligibility greater than 85%, whereas only 10% of individuals with bulbar-onset ALS demonstrated speech intelligibility greater than 85%. Rong et al.15 modeled time to speech loss in individuals with ALS and found that at 2.5 months after diagnosis, 71% of participants with ALS maintained sentence intelligibility scores higher than 85% and 41% of them demonstrated a speaking rate faster than 120 (w/m). At three years after diagnosis, however, only about 20% of their participants with ALS exhibited a speaking rate greater than 120 (w/m) and 30% of them had sentence intelligibility greater than 85%. Differences in study findings can likely be attributed to the fact that their model was based on time from diagnosis (years) rather than time since symptom onset. Considering time from diagnosis may be problematic because diagnostic criteria can vary across clinics and it may, therefore, capture different stages of disease across individuals with ALS. In addition, Rong et al.15 included individuals with ALS irrespective of the site of onset while clinical observations and empirical evidence have consistently indicated that the rate of disease progression and functional decline is significantly more rapid in individuals with bulbar onset than spinal onset44.Because ALS progresses rapidly in some affected individuals, an important goal of clinical management is to predict functional changes in patients’ speech performance in order to facilitate intervention planning, such as the introduction of assistive technology before the patient’s ability to learn these skills is hindered by the severity of their condition45. Our findings further support this assertion, as the slope of change in speech measures was statistically greater than the slope of change in ALS functional measures . The slower rate of decline in clinical measures of ALS function may be expected as a result of the multidimensionality of the ALSFRS-R total score and the ALSFRS-R bulbar subscore, which is different from speech measures that objectively represent performance of participants in a single domain . In addition, the change over time in speech measures was mildly associated with changes in ALS functional measures only for the spinal onset group. This finding is comparable with a study conducted by Barnett et al.18, who reported only mild-to-moderate associations between speaking rate and clinical measures of overall ALS and bulbar disease severity. These findings suggest that ALSFRS-R scores—including the bulbar subscore—do not effectively capture functional decline in speech motor performance over the course of bulbar decline, particularly in individuals with bulbar-onset ALS with greater speech impairment severity and a faster rate of decline in functional speech measures.Research has documented increased sensitivity and responsiveness of instrumental bulbar measures over patient-reported measures of bulbar dysfunction46. In addition, the data used in this study were collected during visits that took place during a relatively confined time window (between 10:00 am and 4:00 pm rather than early morning and late at night) which could minimize the potential confounding effect of “time of day” on speech measures.In sum, this study quantified the rate of decline in functional speech measures controlling for important clinical and demographic factors and provided estimates of time to speech loss in individuals with spinal and bulbar-onset ALS. These findings inform our understanding of speech decline over the course of ALS disease progression and will contribute to the literature supporting best practices for timely intervention. A number of limitations should, however, be acknowledged. First, because of the clinical heterogeneity of ALS, each patient may demonstrate a unique trajectory of speech decline, making prediction within an individual difficult. Although we accounted for factors such as onset site, age at onset, and sex, the models presented in this study only provided simplistic estimates of the rate of decline in functional speech measures of individuals with ALS. Multiple pathophysiological, clinical, epidemiological, and genetic factors singly or in combination, influence the rate of ALS progression. However, producing models of ALS progression that simultaneously account for these variables is almost impractical due to high inter-individual variability in the underlying pathophysiological and clinical manifestations of ALS, heterogeneity in the expression of the disease symptoms, collinearity among predictors, and complex interactions of multivariate factors. Second, the distribution of sex was unbalanced between the ALS-spinal and ALS-bulbar groups, which despite the robustness of LMEs models to unequal sample sizes, might have influenced the findings related to the effect of sex on speech measures of ALS subgroups. Third, we did not track the “time of the day” when the ALSFRS was scored and if that can influence the association between the ALSFRS scores and speech measures. Although we were not able to examine this, it has been reported that “time of day” does not significantly influence speech measures


The novel brain-penetrant peroxisome proliferator-activated receptor gamma agonist leriglitazone, previously validated for other rare neurodegenerative diseases, is a small molecule that acts as a regulator of mitochondrial function and exerts neuroprotective, anti-oxidative and anti-inflammatory effects. Herein, we tested whether leriglitazone can be effective in ameliorating the mitochondrial defects that characterize an hiPS-derived model of Pantothenate kinase-2 associated Neurodegeneration (PKAN). PKAN is caused by a genetic alteration in the mitochondrial enzyme pantothenate kinase-2, whose function is to catalyze the first reaction of the CoA biosynthetic pathway, and for which no effective cure is available. The PKAN hiPS-derived astrocytes are characterized by mitochondrial dysfunction, cytosolic iron deposition, oxidative stress and neurotoxicity. We monitored the effect of leriglitazone in comparison with CoA on hiPS-derived astrocytes from three healthy subjects and three PKAN patients. The treatment with leriglitazone did not affect the differentiation of the neuronal precursor cells into astrocytes, and it improved the viability of PKAN cells and their respiratory activity, while diminishing the iron accumulation similarly or even better than CoA. The data suggest that leriglitazone is well tolerated in this cellular model and could be considered a beneficial therapeutic approach in the treatment of PKAN. Neurodegeneration associated with pantothenate kinase 2 deficiency  is an autosomal recessive disease which belongs to the group of disorders named Neurodegeneration with Brain Iron Accumulation (NBIA) ethoxy] phenyl]methyl]-1,3-thiazolidine-2,4-dione hydrochloride) is the hydrochloride salt of the active metabolite M4 (M-IV) of pioglitazone  in comparison with CoA. We noted that the treatment with leriglitazone did not affect the differentiation of neuronal precursors into astrocytes. Most importantly, it improved the viability of PKAN cells, increased the mitochondrial respiration, and, at the same time, significantly reduced iron accumulation.The hiPS clones for controls and PKAN patients were previously generated and fully characterized . SubsequAstrocytes were treated with leriglitazone  in the range from 3 nM to 3 μM for three days after they reached maturation to determine the optimal dose of the drug. In all the other experiments, astrocytes were differentiated in the presence or absence of 100 nM of leriglitazone or 25 μM of CoA for more than 45 days with fresh medium and drugs changed every 2–3 days.Cells grown on coverslips were fixed in 4% paraformaldehyde and processed for immunofluorescence as described in . Immunof4 astrocytes/well in 96-well plates were grown in the appropriate medium at 37 °C for 18 h and then incubated with 10 μL of MTT solution  for 2 h at 37 °C. The color absorbance was read at 570 nm on an iMark Microplate Reader .A total of 2 × 10The oxygen-consumption rate (OCR) of astrocytes was measured with an XF96 Extracellular Flux Analyzer  as previously described (Orellana).Astrocytes were differentiated for more than 70 days in the presence or absence of 100 nM leriglitazone or 25 μM CoA and then stained for iron content with Perls reaction by incubation for one hour in 1% potassium ferrocyanide, 1% hydrochloric acid in distilled water. Cells were counterstained with nuclear fast red (Sigma). Images were taken on Zeiss AxioImager M2m equipped with AxioCam MRc5 using 40× objective.® software  was used togenerate the graphs and to perform statistical analysis. The results are expressed as mean ± SD or ±SEM. The statistical analysis performed was indicated in the corresponding figure legend. Significant p-values: * p < 0.05; ** p <0.01; *** p < 0.001; **** p < 0.0001 were considered.GraphPad PRISMStarting from the previously obtained hiPS clones , we diffThe differentiation method, previously set up in  and descTo determine the optimal dose for leriglitazone treatment, we added to the growth medium increasing concentrations of the drug ranging from 3 nM to 3 μM and maintained the mature d-astrocytes culture of one control and one patient for 3 days. At the end, we analyzed the d-astrocytes’ viability and respiration, two features affected in PKAN neuronal models ,24, to vBased on these results, we decided to use a concentration of 100 nM of MIN-102 and extend the time of treatment to assess the effect of longer treatment with the drug and directly compare the results with the already proven beneficial effect of 25 μM CoA addition ,26,32, fMaintaining these identified conditions of treatment, we investigated the ability of leriglitazone in the amelioration of the pathological phenotypes. The estimated cell vitality revealed that all the untreated PKAN d-astrocytes were less viable than controls, as expected A. The adRespiratory parameters of PKAN d-astrocytes were also examined by measuring the oxygen consumption rate (OCR) and comparing it with the controls. The results showed that all patient’s cells had significantly lower values of respiratory parameters than controls, showing impaired mitochondrial functionality . Herein,The peculiar feature of this PKAN astrocyte model is the presence of intracellular iron deposits  that areTreatment with CoA was already proven effective in lowering iron aggregation , and herLeriglitazone was recently evaluated in in vitro and in vivo preclinical models of neurodegenerative disorders and clinically tested in healthy volunteers in a phase 1 study ,30 and cHerein, we checked whether this could be confirmed in our PKAN models, which are characterized by mitochondrial dysfunction. Indeed, our data indicated that the treatment of PKAN d-astrocytes during their differentiation with leriglitazone does not affect their proliferation. This could be probably explained by the recovery of the mitochondrial functionality as demonstrated by the rescue of respiratory activity. Interestingly, in two patients, the respiratory activity resulted in higher improvement after leriglitazone treatment compared to CoA treatment, suggesting a similar efficacy of the leriglitazone in rescuing mitochondrial functionality. The positive effect of the leriglitazone treatment cannot be ascribed to the increased amount of the PANK2 enzyme in these cells as seen by the absence of PANK2 peptide in untreated or treated cells. More intriguingly, the phenotype of iron deposition appeared strongly reduced after leriglitazone treatment with a slightly greater efficacy of leriglitazone as compared to CoA in reducing the percentage of cells containing iron deposits. This results is aligned with the data reported recently in FRDA patients treated with leriglitazone in the FRAMES clinical trial, showing that iron accumulation in the dentate nucleus, as assessed by QSM, was greater in the placebo arm . We cannCoA is a key metabolic cofactor for many biochemical functions; among them, it is a vehicle of the acyl group, and it is able to activate the carbonyl groups in many reactions taking place within cells, including the tricarboxylic acid cycle and metabolism of fatty acids . FurtherFurther studies will be needed to define this point; however, these results are interesting from a therapeutic point of view. Until now, PKAN patients have been treated with drugs that alleviate their symptoms. These therapies are based on the usage of dopaminergic drugs, anticholinergics, tetrabenazine, baclofen and botulinum toxin. Other current therapies include the administration of iron chelating agents, such as deferiprone. In recent clinical trials, deferiprone’s long-term effect has been confirmed by the reduction of iron accumulation in the globus pallidus and a stabilization of motor symptoms ,41,42. OC.V., L.R.-P., M.M. and P.P. are employees of Minoryx and have stock options and/or patents with Minoryx.


Ten patients experienced both trientine salts: eight stopped TETA 4 HCL  because this treatment was not available anymore (mean duration 7.4 years). Three of these patients already experienced TETA 2 HCL before the sequence. Two patients with a hepatologic phenotype (one had a previous sequence of TETA 4 HCL before) stopped TETA 2 HCL because of cold storage issues (mean duration 42.8 years). The total number of sequences was 57. All of the patients were clinically stable. No difference in efficacy was detected. Both treatments were well tolerated, except for a case of recurrence of lupus erythematosus-like syndrome in the TETA 2HCL group. The major reason for interruption of TETA 4HCL was due to a discontinuation in production of this salt. The reasons for stopping TETA 2HCL were mainly due to adherence issues largely attributed to the cold storage requirement. Conclusions: The two salts of trientine were effective in treating patients with WD. However, interruption of TETA 2HCL was frequent, linked to the cold storage requirement. As adherence to treatment is a key factor in the successful management of WD, physicians need to be even more vigilant in detecting adherence difficulties in patients receiving treatment with TETA 2HCL.Background: Wilson’s disease (WD) is one of the few genetic disorders that can be successfully treated with pharmacological agents. Copper-chelating agents  and zinc salts have been demonstrated to be effective. There are two salts of trientine. Trientine dihydrochloride salt (TETA 2HCL) is unstable at room temperature and requires storage at 2–8 °C. Trientine tetrahydrochloride (TETA 4HCL) is a more stable salt of trientine that can be stored at room temperature. No comparative study between both of the salts of trientine has been performed to date. As the two chemical forms were available in France between 1970 and 2009, we conducted a study to evaluate their efficacy and safety profiles. Methods: This retrospective cohort study was conducted by reviewing data from the national WD registry in France. Forty-three WD patients who received TETA 2HCL or TETA 4HCL monotherapy for at least one year until 2010 were included. The primary endpoints were hepatic and neurological outcomes. Secondary endpoints were the events leading to a discontinuation of medication. Results: Changes in medication were common, leading to the analysis of 57 treatment sequences of TETA 4HCL or TETA 2HCL. The mean duration of treatment sequence was significantly longer in the TETA 4 HCL group (12.6 years) than in the TETA 2HCL group (7.6 years) ( Wilson’s disease (WD) is an autosomal recessive disorder characterized by pathological copper accumulation in many organs, initially the liver, and then essentially the cornea and brain. It is caused by homozygous or compound heterozygous mutations in the ATP7B gene which encodes a transmembrane copper-transporting P-type ATPase . WD is oCopper-chelating agents  bind with excess copper, forming a stable complex which is excreted mainly in the urine. It has been suggested that trientine salts may also decrease intestinal copper absorption ,4. Zinc ® from Univar, Downers Grove, IL, USA) was used through a compassionate use program for those patients intolerant to D-penicillamine and was only dispensed by hospital pharmacies. This salt is now marketed in the EU as Cufence®, following EU marketing authorization in 2019. Trientine tetrahydrochloride (TETA 4HCL) is a more stable salt of trientine that can be stored at room temperature. In France, TETA 4HCL was available from the mid-1970s until 2009, as a hospital preparation supplied by AGEPS  of the Assistance Publique—Hôpitaux de Paris. This salt was granted a European marketing authorization in 2018 and is currently marketed in Europe as Cuprior® .D-penicillamine is the reference treatment in many European countries, but severe adverse effects are frequent, leading to a discontinuation of this therapy in up to 30 % of patients . The othNo comparative study between both of the salts of trientine (dihydrochloride and tetrahydrochloride) has previously been performed. As the two chemical forms were available in France between 1970 and 2009, we conducted a study to evaluate the efficacy and safety profiles of both of the salts.This study was approved by the Institutional Review Board of HUPNVS, Paris 7 University, AP-HP (n°1343579). An informed consent was obtained from all of the subjects and/or their legal guardian(s). All of the patients signed a written consent form. All of the methods were carried out in accordance with relevant guidelines and regulations, and in accordance with the Declaration of Helsinki.This retrospective cohort study was conducted by reviewing data from the national WD registry. The WD patients who were followed in Lariboisière hospital—Paris  and treated until 2010 were selected. This final date corresponded to the year after which the production of TETA 4HCL by AGEPS was discontinued. The diagnosis of WD was based on clinical symptoms, abnormal copper metabolism and genetic testing with a Leipzig score ≥4 [The different courses of treatment with TETA 4HCL and 2HCL were identified and the treatment sequences of TETA 4HCL or 2 HCL with a follow-up period superior to one year of continuous treatment were analyzed. The events leading to a discontinuation of medication were recorded and classified.The clinical and laboratory data were recorded at the beginning and at the end of each trientine treatment sequence; the duration of the sequence was noted. The patients included in the study were divided into two groups, according to the absence or presence of neurological symptoms. The hepatic assessment included clinical symptoms, measurement of serum transaminase levels, bilirubin and prothrombin time (PT). The presence of cirrhosis  was recorded. The neurological evaluation was based on clinical symptoms. The presence of Kayser–Fleischer rings at slit-lamp examination was documented. The adherence to treatment was recorded by reports of compliance from the patients that were recorded in their medical records.Primary endpoints were hepatic and neurological outcomes. The hepatic outcome was based on clinical symptoms and a course of liver enzymes and liver function tests. The neurological outcome was evaluated by neurological symptoms. Both hepatic and neurologic outcomes were scored as follows: unchanged, improved or deteriorated. The evolution of Kayser–Fleischer rings was also scored as unchanged, improved, disappeared or increased.The secondary endpoints were the events leading to a discontinuation of medication. The reasons for treatment interruption or discontinuation were classified: loss of efficacy, adverse events, treatment non-adherence, manufacturing interruption.The quantitative variables were expressed as median (interquartile range) and the categorical variables as frequencies and percentages. Comparisons between two groups were made using the Student U test for continuous variables and the Fisher exact test for qualitative variables if the frequency was <5, otherwise the chi-squared test was used.From the 248 WD patients recorded in the national registry and who were followed at the Lariboisière hospital before 2010, 62 received at least one sequence of treatment with a trientine salt. Nineteen patients were excluded because they received a zinc salt in combination with trientine over the treatment period. Thus, 43 patients were included in the study . Twenty-Changes in the trientine treatment were common in this cohort. The 43 patients received 57 trientine monotherapy treatment sequences. This included 10 patients who received both TETA 4HCL and TETA 2HCL in different sequences (with a duration of more than one year); 2 patients who received only TETA 4HCl and 31 patients who received only TETA 2HCL. This corresponded to 57 trientine treatment sequences: 13 sequences with TETA 4HCL and 44 sequences with TETA 2HCL .p = 0.011).The mean sequence duration was significantly longer in the TETA 4HCL group, 151.7 ± 111.1 months, vs 91.1 ± 58.1 months in the TETA 2HCL group  ring evolution was comparable in both of the treatment groups. In one sequence of the TETA 4HCL group, a slight increase in the ring was reported, without a deterioration in neurological and hepatic disease .No adverse effects were observed during the TETA 4HCL sequences (mean duration: 12.6 years). All 13 of the TETA 4HCL sequences were stopped during the study period: 11 (85%) due to the fact that manufacturing of the hospital preparation was discontinued, 1 due to difficulties with supply and 1 due to an increase in the Kayser–Fleischer ring, without neurologic or hepatic deterioration.The mean duration of treatment sequence was 7.6 years for TETA 2HCL. At the end of the study period, 26 (60%) of the TETA 2HCL monotherapy treatments were still ongoing. In three patients, zinc was added to TETA 2HCL due to hepatic or neurological deterioration in two cases, and as a result of an increase in liver copper without hepatic deterioration in one case. A bad adherence to TETA 2HCL was suspected in these three cases. The reasons for stopping TETA 2HCL were mainly due to adherence to medication issues (11 cases), generally linked to the requirement for cold storage. Other reasons included one case of lupus erythematosus-like syndrome in a patient with a previous diagnosis of lupus erythematosus during treatment with D-penicillamine, and one liver transplantation for suspicion of hepatocellular carcinoma. Two patients died ; these deaths were not considered as related to WD.®), this study comparing the efficacy and safety of TETA 4HCL to TETA 2HCL in everyday clinical practice is important, since many patients are still taking TETA 2HCL. Efficacy and safety are closely interrelated because any switch in treatment is usually linked to a lack of efficacy, observance issues, adverse effects or difficulties with treatment adherence.Now that TETA 4HCL has received a European marketing authorization and is being marketed in Europe . This switch was mainly due to the occurrence of adverse events. Adverse drug reactions are commonly reported with D-penicillamine treatment and are serious enough to lead to at least 30% of patients on D-penicillamine discontinuing the drug . Four paThe study population is representative of the wide WD population, as it includes children and adult patients. The mean age at diagnosis was 20 years (range 5.6 years to 46.3 years). The majority of the patients presented with liver disease. These data are comparable to those of former studies ,9. AlthoThe majority of the patients either improved or stabilized their symptoms under trientine. No differences in efficacy were detected when assessing the TETA 4HCL and 2HCL treatment sequences for changes in hepatic and neurological symptoms. The parameters relative to hepatic function, in particular serum transaminase levels, tended to improve in both of the groups. Many studies have demonstrated the effectiveness of TETA 2HCL, showing in addition that fewer side effects are observed than with D-penicillamine ,11. In tn = 44), only one adverse event was reported. This was a case of lupus erythematosus-like syndrome in a patient who already had presented with penicillamine-induced lupus erythematosus-like syndrome. However, 11 treatment sequences were stopped as a result of difficulties in treatment adherence due to the refrigeration requirements (often creating problems for patients who needed to work away from home or travel) for TETA 2HCL. The published guidelines indicate that adverse effects are rarely observed with TETA 2HCL treatment: urticaria, reversible anemia and lupus-like reactions are described as the key potential side effects [In this study, the main reason for discontinuation of the drug was the production shutdown of one of the biochemical salts, TETA 4HCL. All 13 TETA 4HCL treatments were stopped due to cessation of manufacturing or supply difficulties, except for 1 patient whose Kayser–Fleischer rings increased. No adverse events were reported in this group following an average treatment duration of 12.5 years. In the TETA 2HCL group ( effects . In our  effects .The number of patients treated with trientine as a first-line therapy is small and does not allow for a satisfactory analysis of this sub-group. However, six other patients started trientine therapy early in the course of this lifelong disease, during the first three months following diagnosis. Trientine was introduced in these patients as a result of early adverse events associated with D-penicillamine. The clinical evolution of these patients was not different to that of the whole cohort.This study has certain limitations, including its retrospective nature and the lack of randomization. The rarity of the disease, the fact that trientine was used in France via a compassionate use program and that manufacturing of TETA 4HCL was discontinued in 2009, explain the low number of patients included in the study. At that time, adherence was subjectively assessed during the medical examination and not with dedicated scores, such as the Morisky score [In conclusion, both of the trientine salts were equally effective in controlling WD. Adverse events were infrequent. In WD, the adherence to medication is a key factor for treatment success. Interruption in the TETA 2HCL therapy was frequent, linked to the requirement for cold storage. The physicians therefore have to be even more vigilant to detect non-adherence to medication as early as possible in the patients being treated with TETA 2HCL.


Severe community-acquired pneumonia (CAP) is a condition that frequently requires intensive care and, eventually, can cause to death. Piperacillin/tazobactam antibiotic therapy is employed as an empiric intravenous regimen, in many cases supplemented with intravenous bolus hydrocortisone treatment. The individual and condition-dependent pharmacokinetic properties of these drugs may lead to therapeutic failure. The impact of systemic inflammation, as well as of hydrocortisone on the altered pharmacokinetics of piperacillin is largely unknown. The protocol of a clinical study aimed at the characterization of the pharmacokinetics of piperacillin and tazobactam and its association with the concentrations of inflammatory markers and adrenal steroids during CAP therapy will be investigated in up to 40 critically ill patients. The serum concentrations of piperacillin and tazobactam, cortisol, cortisone, corticosterone and 11-deoxycortisol and interleukin-6 levels, as well as routine clinical chemistry and hematology parameters will be monitored from the beginning of treatment for up to five days. Nonparametric population pharmacokinetic modeling and Monte-Carlo simulations will be performed to make estimates of the pharmacokinetics of piperacillin and tazobactam and the probability of pharmacokinetic-pharmacodynamic target attainment. The observed individual characteristics and changes will be correlated with clinical and laboratory findings. The protocol of the observational study will be designed following the STROBE guideline. Severe community-acquired pneumonia (CAP) is a leading cause of death among infectious diseases. Up to 20% of adults diagnosed and hospitalized with CAP eventually require intensive care. Of the CAP patients treated at the intensive care unit (ICU), 40–80% require mechanical ventilation, approximately 50% develop severe sepsis or septic shock and the overall mortality is 20–50% ,2,3. TheCAP patients treated in the ICU receive complex parenteral therapy from the first day. The most important components are (1) antibiotics, with piperacillin being commonly employed along with the β-lactamase inhibitor tazobactam, and (2) a steroid, such as hydrocortisone  . Little Additionally, the characteristic of the acute inflammatory phase may also be related to treatment measures since the optimal elimination of the infection requires an adequate initial response of immune system that considers the time and level of extension. Mortality correlates significantly with the magnitude of inflammatory response occurring in CAP, characterized by elevated concentrations of IL-6 and IL-10 . HydrocoPiperacillin is a β-lactam agent effective against several Gram-positive and Gram-negative aerobic and anaerobic strains. It is used in combination with tazobactam, which increases the efficacy of piperacillin and widens its antibiotic spectrum . HoweverInformation on the relationship between piperacillin/tazobactam pharmacokinetics, the adrenocortical function and the clinical status is essential for conducting the personalized therapeutic regime of severe CAP patients at the ICU, which is possibly the key to improving treatment outcomes. We found no clinical studies conducted with the perspective of the complex monitoring of critically ill CAP patients by the simultaneous characterization of antibiotic pharmacokinetics, adrenocortical function, the general clinical status, and routine laboratory parameters. In the current study, we will investigate these relationships to promote the individualization of future treatments.The study protocol was written by the STROBE guideline , thus itThe overall aim of this study is to explore the potential relationships between exposure to piperacillin and tazobactam, the circulating concentrations of adrenal steroids following the administration of hydrocortisone, the activity of the immune system and the clinical status of patients treated with severe CAP (including those developing an acute exacerbation of chronic obstructive pulmonary disease) at the ICU . We willWe sought to accomplish the following objectives:overall objective of the study was to establish a methodology for the evaluation of the consequences of pharmacological intervention as part of the initial phase of the intensive care of CAP patients with the perspective of optimizing treatment outcomes.The Objective 1 is to establish population pharmacokinetic models of piperacillin and tazobactam in critically ill adult patients diagnosed with severe CAP and treated with an intravenous piperacillin/tazobactam preparation, as well as to monitor intra-individual changes in the pharmacokinetic properties within each subject over the first five days of treatment. Additionally, we plan to monitor the changes in concentrations of endogenous steroids during the five-day of treatment and evaluate the possible relationship with pharmacokinetic parameters of piperacillin and tazobactam.Objective 2 is to investigate the chance of successful microbiological cure by performing simulations based on the established population pharmacokinetic models.Objective 3 is to evaluate the concentrations of endogenous molecular biomarkers (glucocorticoids and inflammatory markers), which are supposed to be appropriate to characterize clinical status and to relate them to changes in PK parameters of piperacillin and tazobactam.High inter- and intra-individual variability of PK parameters of piperacillin and tazobactam pharmacotherapy is expected in critically ill patient treated in ICU, which is related to the outcome of treatment.The goal of this study is to test and evaluate this concept by developing a nonparametric population pharmacokinetic model of piperacillin and tazobactam in these specific populations and by comparing the observed changes in the concentration time curves to other clinical, physiological and laboratory parameters.The results of this hypothesis-generating study are expected to provide a better understanding of the relationships between antibiotic pharmacokinetics, hormonal status and the clinical outcome of CAP patients treated at the ICU.This was a prospective, nonrandomized, noninterventional, unicentric and observational cohort study. Patients who meet the eligibility criteria are to be recruited between 1 September 2022 and 15 April 2024 (1.5-year period). The involvement of subjects will begin upon their admission to the Department of Anesthesiology and Intensive Care, Uzsoki Teaching Hospital, and will continue until an exit event occurs, or will be discontinued after day 5. The overview of the study design is presented in We will recruit up to 40 adult patients (age ≥ 18 years) diagnosed with severe CAP and receiving piperacillin/tazobactam and hydrocortisone based on the treatment policy effective at the Central Department of Anesthesiology and Intensive Care, Uzsoki Teaching Hospital Diagnosis. The respective treatment policy has been established based on the international American Thoracic Society and the Infectious Diseases Society of America (ATS/IDSA) 2019 guidelines, according to which the validated definition of severe CAP includes either one major criterion or three minor criteria  22,23].,23.22,23Pseudomonas aeruginosa. The following risk factors of Pseudomonas aeruginosa infection are taken into consideration during diagnosis: chronic obstructive pulmonary disease, chronically persistent home ventilation therapy, status of diabetes mellitus, proven alcohol use disorder, long-term steroid therapy and continuing pharmacotherapy using immunosupressants. Applied antimicrobial prophylaxis before community-acquired pneumonia and registered hospitalization in three months are also connected to clinically relevant assumption to Pseudomonas aeruginosa infection [The inclusion criteria is established as patients with consideration of community-acquired pneumonia clinically suspected to be caused by infectious disease of nfection .Due to the severe condition of the subjects, they will not be approached directly, but the informed consent of close relatives who are legally capable of acting on behalf of the patients will be requested.The study period will be terminated immediately before day 5 concerning subjects who experience any of the following events : (1) death; (2) stage 5 kidney disease requiring renal replacement therapy; (3) discharge from ICU as a result of a remarkable improvement in the subject’s clinical condition; or (4) any other medical events resulting in a special condition that, directly or due to measures regarding its treatment, have an impact on the treatment protocol, on the clinical response to treatment or on the pharmacokinetic properties of piperacillin, tazobactam or hydrocortisone. Data collected before an exit event occurs will be employed for evaluation.Required renal replacement therapy, age < 18 years, state of pregnancy or breast feeding, as well as known intolerance for specified drug components, are defined as applicable exclusion criteria.Subjects who withdraw their consent to participate will also be excluded immediately without any negative impact to their care or other services.The decision to terminate the investigation of a subject  or to exclude a subject will be made by the principal investigator (Cs. K.).Based on the therapeutic indication, enrolled subjects will be administered 3-h intravenous infusion containing 4 g piperacillin along with 0.5 g tazobactam every 6 h (q6h). No bolus dose of piperacillin/tazobactam will be administered at the beginning of treatment. Intravenous injection of 50 mg hydrocortisone will be applied q6h. Other therapeutic measures will be applied according to effective international and local protocols guiding the treatment of severe CAP patients at the ICU.Blood and tracheal secretion will be collected from subjects. Blood samples will be drawn via the cannula inserted into the vena cava superior strictly based on the relevant therapeutic protocols. The issue corresponding to contamination of the samples with flushing liquid  is addressed by discarding 3 mL of solution (mixture of blood and flushing liquid) before blood sample is collected for investigation.While the femoral vein could also be accessed, its location  may result in samples which would be different concerning their composition. No risks other than those associated with blood sampling employed routinely as part of the provided intensive care have been identified. Tracheal secretions will be collected through the endotracheal tube by inserting a suction catheter connected to a sputum trap .The planned sampling scheme is shown in The susceptibility of the pathogen CAP to piperacillin will be evaluated by collecting tracheal secretions before starting the antibiotic treatment, and assuming that no exit event occurs, on day 4 of the treatment, and by establishing the MIC of piperacillin/tazobactam.A series (15) of two-fold dilutions of an antibiotic are incorporated on a plastic carrier strip . From the strip antibiotic agent could diffuse freely into the Mueller–Hinton agar to generate a diffusion gradient along the length of the strip. After incubation the MIC could be defined as the point where the growth inhibition ellipse intersects the MIC scale on the strip. Turnaround time of the measurement could differ depending on the patient specific clinical status but generally it is conducted in 48 h .To evaluate the serum concentrations of piperacillin and tazobactam, 6 native venous blood samples with a volume of up to 3.5 mL will be collected into vacutainer tubes on each of the first 5 days of antibiotic treatment at the following relative times with respect to the initiation of the daily first intravenous piperacillin/tazobactam infusion: 3.25 h; 3.5 h; 4 h; 4.5 h; 5 h; and 5.5 h. Following their collection, samples will be centrifuged immediately. Separated serum will be transported in a 2.0-mL polypropylene microcentrifuge tube for further processing to the Central Laboratory of Uzsoki Teaching Hospital.Aliquots B/1-1–B/1-6 : In total, 100 µL serum will be separated from samples ‘B/1-1′–‘B/1-6′ and 20 µL Chromsystems® Antibiotics with HPLC Priming Solution (cat. 61012) will be added. The mixture will be vortexed and stored at −70 °C until analysis. Serum concentrations of piperacillin and tazobactam will be measured using high performance liquid chromatography with absorbance detection (HPLC-UV) using Chromsystems® Antibiotics in serum/plasma with HPLC reagent kit with a CE-IVD certificate  [® kit. The protein binding was found to be about 30% for both drugs [Hungary) . Total c cat. 610 will be Aliquot B/2 : In total, 250 µL serum will be separated from sample ‘B/1-6′ and stored at −70 °C until analysis. These aliquots will be prepared from the 5.5-h samples collected after the initiation of the first piperacillin/tazobactam infusion on each day. The daily first infusion of piperacillin/tazobactam will be applied between 7.00–9.00 a.m., and aliquot ‘B/2′ will be obtained in a 2-h interval (12.30–2.30 p.m.). Consequently, the circadian rhythm of the molecules of interest is expected not to have an appreciable impact on the results.The aliquots will be stored at −70 °C until analysis. Up to 5 Aliquots B/2 will be collected from each subject. Cortisol, cortisone, corticosterone and 11-deoxycortisol will be assayed using ultra-high performance liquid chromatography and tandem mass spectrometry (LC-MS/MS) using a validated in-house method . SamplesHere, 1 tube (8 mL) of venous native blood will be collected to perform interleukin-6 (IL-6) and routine clinical chemistry assays on each day of treatment prior to the administration of the first piperacillin/tazobactam infusion. Samples will be centrifuged within 2 h. A maximum of 5 of these samples are going to be donated by each subject.Aliquot C/1 (IL-6): In total, 1 mL serum will be separated and stored at −18 °C until analysis. using a Roche Cobas e601 analyzer with Roche Cobas Elecsys IL-6 reagent . Samples will be stored at −70 °C for 1 year following analysis.Aliquot C/2 : In total, 3 mL serum will be separated and assayed on the same day using a Roche Cobas Elecsys Brahms platform for assaying procalcitonin. The other tests  will be performed on a Beckman Coulter analyzer . Samples will be stored at 2–8 °C for 2 days following analysis.Here, 1 tube (2 mL) of heparinized blood from radial artery will be collected for determining blood pH, lactate, partial oxygen pressure and arterial oxygen saturation before the administration of the first piperacillin/tazobactam infusion on each day of treatment. Analysis will be performed within 10 min after collection using an ABL800 Flex Blood gas analyzer . Up to 5 of these samples will be collected from each participant and destroyed upon analysis.3-EDTA) to perform complete blood count analysis. Analysis will be performed within 4 h on Cell Dyn Sapphire and Cell Dyn Ruby analyzers . A maximum of 5 samples during the study will be obtained from each subject. Samples will be stored at 2–8 °C for 2 days following analysis.Next, 4 mL venous blood will be collected before the administration of the first piperacillin/tazobactam infusion on each day of treatment into a tube containing tripotassium ethylene diamine tetraacetate . A maximum of 5 samples will be obtained from each subject. Plasma samples will be stored at 2–8 °C for 2 days following analysis.At the end of the storage period, samples will be treated as hazardous samples and disposed of in accordance with the applicable regulations.No intervention other than those performed as part of the treatment of recruited subjects guided according to the effective therapeutic protocols will be initiated. The collection of tracheal secretions as described here is part of the treatment. Blood samples will be drawn via a cannula inserted in the vena cava superior as a therapeutic measure. The collection of blood samples will not cause any discomfort or present any risks to subjects. Samples C, D and E are collected as described from all CAP patients receiving intensive care as part of their treatment. Samples B are going to be obtained for the purposes of the study.Following admission to the ward, severity scores are going to be established to predict treatment outcomes, including simplified acute physiology score II (SAPS II), sequential organ failure assessment (SOFA), mortality probability model (MPM), confusion, yremia, respiratory rate, blood pressure, age ≥ 65 years system (CURB-65), and Pneumonia Severity Index (PSI) ,30,31,32Data will be obtained from patient records  and stored electronically in a spreadsheet format using Microsoft Excel .The minimum acceptable observational set is one complete set of daily samples. Additional issues corresponding to missing values will be addressed by applying the most suitable statistical strategy including, possibly, the exclusion of the variable, the imputation of the mean or median values or if inevitable the omission of the subject from the evaluation. Missing drug concentrations will not be replaced by calculated values.The different variables will belong to one of the following categories: primary or secondary outcome, descriptive, or predictive . PrimaryNonparametric statistical hypothesis tests will be applied to test the relationship between the values of descriptive and predictive variables, and primary and secondary outcomes. Due to the relatively small size of the cohort, this approach is expected to yield more valid results than those obtained using parametric tests following the transformation of data. Bi- or multivariate logistic regression analysis will be employed to create novel predictive scores based on the values of the observed and calculated variables. For each variable that remains in the model, the odds ratio will be calculated. Wald statistics will be employed to select the independent variables associated with the outcomes of interest. Receiver operating characteristic (ROC) curves of the predictive scores will be constructed to characterize their values in predicting the secondary outcome  . The areAssay error equations of piperacillin and tazobactam are going to be constructed by calculating the standard deviations of measured concentrations in 3 independent experiments at a total of 20 spiked concentrations levels, and by using 20 independent serum matrices, which do not contain piperacillin or tazobactam, at each spiking level. Based on existing evidence, Theil’s regression will be performed on the concentration-standard deviation data pairs to establish the error equations ,35.Nonparametric population pharmacokinetic modeling and Monte-Carlo simulation are going to be conducted using PmetricsTM, a dedicated software package running in the R environment ,37,38. OPrimary endpoints: (1) PK parameters of piperacillin and tazobactam and (2) the possible changes in concentrations of the monitored endogenous steroids during the five treatment days.Secondary endpoints: Based on Monte-Carlo simulation the probability target attainment (PTA) of piperacillin and tazobactam.Tertiary endpoints: the association between individual pharmacokinetic parameters of piperacillin and tazobactam and individual laboratory parameters that characterize physiological status.Our work is intended to be a hypothesis-generating observational study yielding preliminary results that could be the basis of hypothesis-driven clinical investigations. In a report of CAP cases from 2016 to 2020 at Central Department of Anesthesiology and Intensive Care  122 patients with CAP were registered (30 patients per year). Wallenburg et al. conducted a clinical study with similar objectives in which work 39 patients were enrolled [Additionally, the sample size was characterized to be suitable for considering the objectives of the study and with the intention to allow the formation of subpopulations during the evaluation of results.All therapeutic measures will be taken based on the clinical protocols applied at Uzsoki Teaching Hospital. Therefore, no specific adverse events other than the minimal discomfort caused by the collection of samples B are expected to occur.The study was approved by the National Institute of Pharmacy and Nutrition  and by the National Scientific and Ethical Committee of the Medical Research Council  (permission number: 261-IK/2020). The written informed consent of included subjects or, if they are incapacitated, of their relatives, will be obtained in advance. All information on subjects is treated anonymously for the purposes of the study. De-identification is performed by the study coordinator. The decision of participants or the persons acting on their behalf to exit the study will be effected immediately regardless of the underlying reason, and without any further adverse impact on their treatment. All provisions of the Declaration of Helsinki of the World Medical Association will be respected and complied with.Standard scoring systems generally employed to describe the clinical condition and perspectives of subjects are going to be applied. Demographic data will be presented as medians and interquartile ranges. Pharmacokinetic model outputs, as well as the probabilities of target attainment are going to be provided in a graphical and tabular format as appropriate. To assess the relationship between candidate covariates and random effects, correlation plots and equations will be presented. The results of nonparametric hypothesis tests performed on unpaired data will be displayed along with boxplots. Covariate exploration will be conducted by comparing the performance of the respective hierarchical (nested) models.The data that support the findings of this study will be openly available in Mendeley Data at doi:10.17632/w7gt8zdp3r.1.The first limitation of the planned study is that, given the fact that the relatively small number of patients admitted to ICU where the study will be conducted leaves little room for applying specific exclusion criteria. The clinical and physiological status of the included subjects is difficult to predict, consequently, the studied population is expected to be heterogeneous in terms of the studied variables. Second, the sampling and evaluation protocols are rather complex. Proper training and supervision of the personnel of the participating ICU will be executed to facilitate correct sample collection and documentation at the point of care.Several samples and aliquots will be obtained from each subject which poses a major logistic challenge. We aim to overcome this difficulty by training the participating laboratory personnel on a regular basis  to assist them in performing their study-related tasks efficiently, by providing continuous supervision of the processes of the preanalytical phase by a laboratory professional, and by enforcing extensive documentation of all procedures.Piperacillin and tazobactam are chemically unstable molecules, therefore, the proper preanalytical handling of samples B/1 is a key prerequisite for obtaining valid pharmacokinetic information. Training, supervision and extensive documentation of related processes will be affected carefully.To our knowledge, the planned study will be the first to characterize the relationship between the antibiotic and endogenous steroid concentrations during the administration of piperacillin/tazobactam and hydrocortisone to CAP patients treated at the ICU. Such therapies are associated with the high inter- and intra-individual variability of PK parameters , but are