key: cord-0954433-it0t7zfj authors: Umbrello, Michele; Guglielmetti, Luigi; Formenti, Paolo; Antonucci, Edoardo; Cereghini, Sergio; Filardo, Clelia; Montanari, Giulia; Muttini, Stefano title: QUALITATIVE AND QUANTITATIVE MUSCLE ULTRASOUND CHANGES IN COVID-19 RELATED ARDS PATIENTS date: 2021-08-15 journal: Nutrition DOI: 10.1016/j.nut.2021.111449 sha: d835e77ececcaf11a867f6c786a46c364dcfadf8 doc_id: 954433 cord_uid: it0t7zfj BACKGROUND & AIMS: Severe forms of COVID-19 are associated with systemic inflammation and hypercatabolism. We compared the time course of the size and quality of both rectus femoris and diaphragm muscles between critically-ill, COVID-19 survivors and non-survivors, and explored the correlation between the change in muscles size and quality with the amount of nutritional support delivered and the cumulative fluid balance. METHODS: Prospective observational study in the general ICU of a tertiary care hospital for COVID-19. The right rectus femoris cross-sectional area and the right diaphragm thickness, as well as their echodensities were assessed within 24 hours from ICU admission and on day 7. Anthropometric and biochemical data, respiratory mechanics and gas exchange, daily fluid balance and the amount of calories and proteins administered were recorded. RESULTS: 28 patients were analysed (age 65±10 years, 80% males, BMI 30.0±7.8). Rectus femoris and diaphragm sizes were significantly reduced at day 7 (-26.1 [-37.8;-15.2] and -29.2 [-37.8;-19.6]%, respectively) and this reduction was significantly higher in non-survivors. Both rectus femoris and diaphragm echodensity were significantly increased at day 7, with a significantly higher increase in non-survivors. The change in both rectus femoris and diaphragm size at day 7 was related to the cumulative protein deficit (R=0.664, p<0.001 and R=0.640, p<0.001, respectively), while the change in rectus femoris and diaphragm echodensity was related to the cumulative fluid balance (R=0.734, p<0.001 and R=0.646, p<0.001, respectively) CONCLUSIONS: Early changes in muscle size and quality seem related to the outcome of critically-ill, COVID-19 patients, and be influenced by nutritional and fluid management strategies. Ever since the novel coronavirus disease (COVID-19) has been declared a global pandemic more than 125 million confirmed cases and more than 2.5 million confirmed deaths were recorded globally [1] . While most cases are characterized by minimal flu-like constitutional symptoms or may even be asymptomatic, some patients develop a severe pneumonia which may lead to acute respiratory distress syndrome (ARDS), multi-organ failure, and death [2] . Systemic hyperinflammation and a procoagulant state play a major pathophysiological role in these severe forms [3] . The increased glucocorticoid and catecholamine production are the main factors driving hypercatabolism and anabolic resistance [4] . Catabolism from muscle proteins, muscle weakness, and/or atrophy, have all been linked to increased morbidity and mortality [5] . Moreover, patients with inadequate energy and protein intake are at risk for complications and negative outcomes [6] . Adequate reserves of body protein at admission to the ICU may then be crucial to recovery and survival, as well as the degree of muscle wasting during the acute phase of the disease. Even more than other categories of critically ill patients, those admitted to the ICU for severe cases of COVID-19 may accumulate nutritional deficits during their first days of ICU stay, which may then play an important role in ICU and hospital outcomes, including mortality and acquired infections [7] . Recent studies showed how feeding intolerance is common in these patients [8, 9] , a finding which cannot be solely explained by the deranged gas exchange, the effects of vasoconstriction from vasopressor or the elevated doses of sedatives and opioids required to facilitate mechanical ventilation [10] . From a nutritional perspective, it has been shown that reaching both protein and energy targets in critically ill patients had a positive impact on survival [11] . The early caloric deficit has already been showed as an independent predictor of ICU mortality together with SOFA score, male sex, obesity and diabetes, suggesting that any effort should be made to implement timely and adequate nutritional support during the ICU stay [12] . Ultrasound is increasingly being used to assess both changes in muscle size and quality over time [13] . Muscle ultrasound has gained wide acceptance as a tool to assess and track changes in muscle structure and composition, potentially improving classification of patients who may be at risk of muscle wasting. Sonography may in fact allow for the assessment of both muscle mass (thickness or cross-sectional area) and quality (echodensity) [14] . In particular, it is well known how both diaphragm and limb muscle size, structure and function deteriorate during the course of ICU stay; sonographic findings of reduced diaphragm thickness and rectus femoris cross-sectional area have been found to be associated with poor clinical outcomes [5, 15, 16] . Similarly, changes in muscle echodensity, easily assessed using grayscale analysis of ultrasound images, have been associated with negative outcomes [17, 18] . Few studies investigated at the same time the change in respiratory and limb muscle size and quality in critically ill patients [19] [20] [21] , and none, to the best of our knowledge, did so in relation to the outcome of patients suffering from COVID-19 related acute respiratory failure. The main aim of this observational study was to compare the change over the first week in the size and quality of both rectus femoris and diaphragm muscles between critically ill, COVID-19 patients who did and did not survive their ICU stay. Secondary outcomes were to explore possible correlations between the change in both muscles size and quality with the amount of nutritional support delivered and the cumulative fluid balance. Ethical approval for this study (Registro Sperimentazioni n. 2020/ST/207) was provided by the Comitato Etico Interaziendale Milano Area 1. Written informed consent was obtained according to Italian regulations All consecutive patients admitted from November 15 th to December 30 th 2020 to the general ICU of a tertiary care hospital were considered for enrolment. Inclusion criteria were age ≥18 years, admission for acute hypoxemic respiratory failure, undergoing invasive mechanical ventilation for no longer than 48 hours and with confirmed SARS-CoV-2 infection. Exclusion criteria were being under 18 years, pregnancy, trauma to the right lower limb, history of neurological, neuromuscular or muscular wasting disease and prolonged immobility prior to admission to the ICU. Within 24 hours from ICU admission, patients underwent diaphragm and rectus femoris ultrasound. The same measurements were repeated at day 7 if the patient was still in the ICU. Anthropometric data, biochemical parameters of inflammation and organ function, respiratory mechanics and gas exchange data were collected. Fluid balance and the amount of calories and proteins administered were recorded on a daily basis All patients were deeply sedated and mechanically-ventilated at ICU admission. The clinical management of patients was standardized according to local and regional suggestion [22] . In particular, all patients received muscle relaxants during the first week of ICU stay, as well as a 10-day course of i.v. dexamethasone 6 mg/day [23] . No other immunomodulatory agents were administered, nor were antiviral drugs. As far as the nutritional support is concerned, ESPEN guidelines for management of COVID-19 patients were followed, and hypocaloric nutrition (about 70% of estimated energy expenditure) was provided in the early phase of acute illness, with increments up to 80-100% between day 3 and 7. As indirect calorimetry was not available, energy and protein targets (25 kcal/kg/day and 1.3 g/kg/day, respectively) were calculated according to real or adjusted body weight, as needed [24] . When no absolute contraindications were present, high-protein enteral feeding (Nutrison Protein plus, Nutricia International B.V., Hoofddorp, NL) was started within 48 hours of ICU admission, initially at a rate of 20 mL/h and increased until the desired goal was reached. Enteral feeding was administered via nasogastric tube; a postpyloric access was positioned in case of gastric intolerance despite prokinetic therapy. Calories contained in propofol or glucose solutions, and the use of supplements were accounted for when total calorie and protein delivery were calculated. Adequacy of calories and protein received was expressed as a percentage of the prescribed nutrition. Data on the onset of symptoms, medical history and current medications at time of symptoms onset, clinical and laboratory data at admission, treatment data, and outcome were collected. Severity scores (simplified acute severity score -SAPS II-and sequential organ failure assessment -SOFA) were calculated at admission. The clinical frailty scale was used to summarize the overall level of fitness [25] . Anthropometric measurements of body length and weight were recorded at ICU admission. Actual body weight was obtained by weighing beds when available, or it was defined as the weight reported by the patient immediately prior to ICU admission, or from information obtained by the families. Ideal body weight was defined as the weight based on the patient's height calculated to a BMI of 25 kg/m 2 . Adjusted body weight, which was used as the reference weight for nutritional targets in the obese patient, was calculated as (actual body weight -ideal body weight) * 0.33 + ideal body weight [26] . Fluid balance was evaluated daily during ICU stay. Fluid intake included intravenous fluids, total parenteral and enteral nutrition, blood products, and intravenous medications. Fluid output included urine, faeces, blood loss, output from drains and other body cavities, gastric aspirate and respiratory evaporation [27] . All variables were derived directly from the computerized clinical records (Digistat Intensive Care Unit, Ascom, Baar, CH). The cumulative fluid balance was the sum of the daily balances. As an index of adequacy of the nutritional support, the cumulative energy and protein deficit were calculated, as the difference between the calorie or protein target and the amount actually delivered to the patient [28] . The right rectus femoris muscle and the right diaphragm were assessed for muscle size and echodensity within 24 hours from ICU admission and then on day 7. Ultrasound device settings, depth and gain were kept constant using the same image presets between patients, as previously described [29] . Ultrasound was performed by a single, experienced operator (MU, Staff Intensivist, >10 years of muscle ultrasound experience). B-mode images were obtained utilizing a 6-14 MHz linear array on a Esaote MyLab X8 device (Esaote SpA, Genova, Italy) with the patient at 30 degrees for diaphragm measurements [30] and in the supine position for rectus femoris ultrasound [31] . End-expiratory diaphragm thickness was assessed in the zone of apposition of the diaphragm to the rib cage. The linear probe was placed above the right 10 th rib in the midaxillary line, as previously described [32] (Figure 1, panel B) . Cross-sectional area of the rectus femoris was measured with the rested leg supported in passive extension. B-mode ultrasonography using the same linear transducer array was applied, similar to the method previously described [13] . Briefly, the probe was placed three-fifths of the distance from the anterior superior iliac spine to the superior patellar border, transversely to the ( Figure 1 , panel A). Since muscle and subcutaneous fat can easily be compressed, a minimal amount of pressure was applied on the tissue under an ultrasound probe sufficiently covered with gel, in order to optimize imaging conditions. To improve ultrasound reproducibility, we temporarily marked and regularly reinforced the skin landmarks. The cross-sectional area was outlined by a movable cursor on a frozen image and calculated. The methods for image acquisition and analysis of both muscles have previously shown good to excellent reliability [33] . All the measurements are reported as the average of three consecutive measurements within 10%. Images were saved in JPEG format and echodensity was quantified using a greyscale histogram analysis of the images, with values ranging between 0 (black) and 255 (white). The analysis was performed with ImageJ software (https://imagej.nih.gov/ij/index.html; NIH, Bethesda, USA). The hand-free tool was used to define the largest free-form area devoid of artifacts; a grayscale frequency histogram was then generated for the selected region, and the median value was recorded [34] . The assumption is that the higher the average density of a muscle region of interest, the lower is its quality (i.e. more intramuscular fat, edema or connective tissue). Images were reviewed by a second investigator (LG) who was not directly involved in image acquisition. Figure 1 shows the muscle ultrasound images at ICU admission and on day 7 from a representative patient. Reproducibility was assessed by building a Bland-Altman plot and calculating the intra-class correlation coefficient [35] : admission images were assessed a second time by the same investigator for intra-observer reproducibility, while day 7 images were analysed by a second, independent investigator for inter-observer reproducibility. Given the lack of similar investigations in COVID-19 patients, a priori sample size calculation was not performed; the sample size was pragmatically based on a 2-month time frame as well as recently published literature on similar topics [17, 18] . Patients with only one ultrasound measurement (i.e. those discharged alive or who died before the 7 th day) were excluded from the analysis. We compared the variables in patients who did and did not survive the ICU stay. Comparisons between normally distributed variables were performed by Student's t test, while non-normally distributed variables were compared by Wilcoxon signed rank test. Normality was tested by the Shapiro-Wilk test. Normally distributed data are indicated as mean±SD, while median and interquartile range are used to report non-normally distributed variables. Association between two variables was assessed by linear regression. The comparison between survivors and non-survivors was performed by analysis of variance for repeated measurements, with time as a within-subject factor and the outcome as a fixed, between-subject factor. The model included the interaction effect of time on the outcome. The statistical significance of the within-subject factor was corrected with the Greenhouse-Geisser method. Pairwise, post-hoc multiple comparisons were carried out according to Tukey method. The statistical analysis was carried out with STATA version 14.0 (Statacorp, College Station, TX, USA); two-tailed Pvalues <0.05 were considered for statistical significance. 36 consecutive patients were enrolled in the study. 8 patients died before day 7; ultrasound images at admission and after 7 days were available for all the remaining 28 patients. Supplementary figure S1 shows the diagram of patient flow during the study period. ICU: Intensive care unit. White circles represent energy intake in ICU survivors, black circles represent energy intake in nonsurvivors; white squares represent protein intake in ICU survivors, and black squares represent protein intake in ICU nonsurvivors. The analysis was performed with factorial analysis of variance, see the methods section for further details. Figure 2 shows the time course of energy and protein intake during the first 7 days of ICU stay in survivors and non-survivors. Energy intake significantly increased from admission to day 7 (p<0.0001), with no statistically significant differences between the groups (p=0.1482). On the other end, protein intake, which also significantly increased over time, was higher in survivors (p<0.0001 for both factors). Calorie intake from propofol ranged from 25.6 to 33.3% of total daily calories in survivors and from 24.6 to 40.0% in non-survivors (p=0.4550). Muscle ultrasound was performed in all patients. Figure 3 shows the evolution of muscle mass (RF CSA and diaphragm thickness) and echodensity over time. The intra-class correlation coefficient for intra-rater The size and echodensity of rectus femoris and diaphragm muscles at ICU admission and on day 7 in survivors and non-survivors is shown in table 2. A significant reduction in the rectus femoris cross sectional area was recorded after the first 7 days of ICU stay. Moreover, the cross-sectional area was lower in non-survivors. Similar findings were seen for the end-expiratory diaphragm thickness. Both rectus femoris and diaphragm echodensity significantly increased during the first 7 days of ICU stay, and in both cases non-survivors had a significantly higher echodensity. Table 3 shows the nutritional parameters on day 7 as well as the changes in muscle ultrasound size and echodensity from admission to day 7 in survivors and non-survivors. No differences were found in urine nitrogen output or serum prealbumin levels. Moreover, the cumulative energy deficit over the first week of ICU stay was not different between the groups. On the other side, the cumulative protein deficit was significantly higher in ICU non-survivors, as was the cumulative fluid balance. The reduction in both rectus femoris and diaphragm size from baseline to day 7 was significantly greater in non-survivors, as was the increase in echodensity. (Figure 4 ). To the best of our knowledge, this is the first report to investigate the time course of both respiratory and limb muscle mass and quality in critically ill patients suffering from COVID-19, and to relate the findings with the nutritional strategy and the outcomes. The main findings of this prospective, observational study are: 1) both rectus femoris cross-sectional area and diaphragm thickness are significantly reduced after one week of ICU stay and this phenomenon is significantly higher in ICU non-survivors; 2) both rectus femoris and diaphragm echodensity are significantly increased after the first week of ICU stay with a significantly higher increase in ICU non-survivors; 3) the decrease in both diaphragm and rectus femoris size is significantly related to the protein deficit over the first week; 4) the increase in both diaphragm and rectus femoris echodensity is significantly related to the cumulative fluid balance over the first week of ICU stay. One of the aims of nutritional support is to potentially mitigate the loss of lean tissue during a state of hypermetabolism and catabolism and consequently improve patient outcomes. Significant muscle wasting occurs early in critically ill patients [36] . The underlying mechanisms, despite being still under investigation, are likely multifactorial and include altered substrate metabolism, anabolic resistance, hypoxia, inflammation, immobilization, and nutritional inadequacy [37] . Notably, all of the former have been described in severe cases of COVID-19. A recent study on critically-ill patients with COVID-19-related acute respiratory failure showed how even achieving the supposed nutrition targets, the nitrogen balance remained negative by on average -9 grams of Nitrogen/day over the first week of stay [4] . Moreover, a recent prospective investigation showed how only about 70% of critically ill COVID-19 patients reached their caloric target on day 4, whereas less than 25% reached their protein target by the same day [38] . A recent longitudinal investigation showed how severe and critical COVID-19 patients showed a 30% reduction in rectus femoris cross-sectional area with an average 16.8% increase in echodensity from days 1 to 10 [39] . We confirmed an early and rapid wasting of both respiratory and limb muscles in patients suffering from COVID-19 related acute respiratory failure, which was even more pronounced in those patients who eventually went on to die. Moreover, despite a protocolized nutritional strategy, calorie intake was not different among groups (likely because of the significant amount of energy provided as propofol infusion), whereas the nutritional intake of proteins was lower in non-survivors. Nitrogen output on day 7 suggest a similar catabolism in the two groups, which may reflect the contribution of an unintended lower protein intake in non-survivors. Indeed, the observational nature of our study precludes any inference about the causal nature of this association. In humans, COVID-19 causes anorexia, myalgias and muscle loss. SARS-CoV-2 spike protein uses the angiotensinconverting enzyme 2 (ACE2) receptor to bind to and subsequently enter the cells. Skeletal muscle cells express an ACE2-receptor which might in part explain the muscle symptoms. This, together with immobility and invasive mechanical ventilation, can lead to a severe form of sarcopenia during the acute phase of COVID-19 [40, 41] . Indeed, diaphragm and limb muscles are known to be different in their composition, as different is their susceptibility to a given injury [42] . We chose to focus on the rectus femoris as it was shown that lower (but not upper) limb muscle thickness and architecture of undergo early rapid changes after ICU admission, potentially reducing force generation and contributing to ICU-acquired weakness [43] . Skeletal muscle injury was also reported to be directly associated with SARS-COV-2 infection [44] . As far as the diaphragm is concerned, a wide array of factors, among which mechanical ventilation and suppression of spontaneous inspiratory effort [15, 45, 46] were shown to lead to an early and acute diaphragm injury in critically ill patients. Indeed, in patients with severe forms of COVID-19 undergoing invasive mechanical ventilation, a large and prolonged use of muscle relaxants has been described [47] . We speculated that patients with COVID-19 related acute respiratory failure might be at significant risk of diaphragm and limb muscle wasting, and we sought to assess the association of such wasting with the nutritional strategy and the outcome. To do so, we used quantitative muscle ultrasound, which is a non-invasive, reproducible and relatively inexpensive imaging modality that was shown to facilitate the assessment of muscle mass and quality based on tissue composition [13, 14] . We found that a significant reduction in muscle mass and change in muscle structure occurred in all patients; such changes were more pronounced in non-survivors and were significantly related to the cumulative protein deficit and the fluid balance. Despite the sample was composed of relatively young males with few comorbidities and in an overall "fit" state as assessed by the low comorbidity index and clinical frailty scale, the results of the current study confirm the rapid and significant deterioration in skeletal muscle size and quality as compared to previous studies in unselected ICU patient groups [5, 17, 36, 48] . We observed an average decrease in rectus femoris cross-sectional area and diaphragm thickness of 27% and 29%, respectively, with even higher decreases in non-survivors. Quite interestingly, we were unable to find any difference between the groups in the other factors that are known to impact muscle mass during ICU stay, such as glycemic control, vasoactive drugs, aminoglycoside antibiotics. In other studies, even smaller changes were associated with physical function at ICU discharge [49] , in-hospital mortality, length of mechanical ventilation and development of ICU-acquired weakness [50] . Several previous studies have shown decreases of diaphragm thickness during the early course of mechanical ventilation, associated with an impairment in diaphragm function and a poor clinical outcome [51] . At least a part of mechanically-ventilated patients experiences early rapid increases in diaphragm thickness, which themselves predicted prolonged ventilation, raising the possibility of a clinically significant diaphragm injury caused by insufficient respiratory muscle unloading during ventilation [15] . In the current investigation, we were unable to find any patient who increased their baseline diaphragm thickness, likely because all of them were undergoing controlled mechanical ventilation and receiving muscle-relaxant agents, then excluding the chance of overuse myotrauma. Consistent with previous reports, non-survivors had at admission a lower size and a higher density in both the diaphragm and the rectus femoris. Previous investigations showed how both a low skeletal muscle area or density, as assessed by CT scan, upon ICU admission are associated with an increased mortality, independent of the severity of disease [52, 53] . In the current study, muscle mass and quality was assessed using ultrasound; indeed, rectus femoris echodensity, as a marker of muscle quality, increased over the first week by on average 12%, which is similar to prior published data [16, 17, 31, 48, 49] . More recently, changes in diaphragm echodensity during the early course of mechanical ventilation were described and associated to a negative outcome [18] . In both cases, the changes increments in muscle echodensity are presumed to be the consequence of either inflammation or infection, or edema due to fluid shifts in the context of volume resuscitation with a positive fluid balance [54] . Interestingly, we found that diaphragm echodensity did not change from baseline in patients who eventually went on to survive, which could in theory suggest a recovery of an initial early muscle injury. As a matter of fact, due to the stress imposed on the healthcare system by the wave of the big surge in the number of COVID-19 patients, several patients underwent prolonged non-invasive ventilator assistance while waiting for an available ICU bed, and we cannot exclude the development of underassistance myotrauma from insufficient ventilator support before intubation [55] . Several studies found associations between protein intake and improved outcomes, at least in specific subgroups of patients such as those at high nutritional risk or with normal kidney function [56] [57] [58] . We found an association between a negative protein deficit and a larger reduction in muscle mass, which may potentially be the link between protein administration and improved outcomes. Indeed, an early higher protein intake was associated with lower mortality in patients with low skeletal muscle area and density but not in patients with normal skeletal muscle area [59] . A low muscle area (that is, a low muscle mass) is considered a proxy for low protein reserves, while low muscle density is associated with qualitative changes such as muscle inflammation and fatty infiltration, which may in turn create an environment of low-grade inflammation and insulin resistance, thereby contributing to anabolic resistance. Identifying these patients might improve risk-stratification and help guide treatments; however, CT-scan assessment of muscle mass and quality is limited by its logistic and health-related risks. We have shown how the ultrasound evaluation of muscle mass and quality might provide clinically meaningful information, with the certain advantages of a bedside-available, repeatable, safe, low-cost procedure. Notably, and similar to previous investigations [53, 60, 61 ] ICU non-survivors had a significantly higher positive cumulative fluid balance as compared to survivors, as well as a cumulative negative protein deficit. In an effort to investigate the causes of such difference, we measured the nitrogen output on day 7, which was not statistically different between the groups. From this, we inferred that the reason for a protein deficit was not the increased loss but a reduced intake. In fact, the amount of proteins administered with the nutritional support was significantly lower in non-survivors (see figure 2 ). One possible explanation for such a reduced protein administration might be a higher incidence of intolerance to enteral feeding in non-survivors, which is known to be a marker of a more severe disease state and associated with a worse outcome, especially in the COVID-19 population [62] . No differences were found in the cumulative energy (calorie) deficit, likely because a significant part of the calorie provision came from intravenous lipids from propofol infusion. Similar findings were recently reported in a population of surgical critically-ill patients [28] . The rationale behind the negative impact of a cumulative protein deficit may depend on a decreased immunocompetence, an increased skeletal muscle catabolism, and impaired wound healing. As a matter of facts, several recent investigations support the increasingly appreciated importance of protein in improving survival [53, 56] . The exact amount of protein requirement in critical illness remains controversial, especially since this is generally scaled upon body weight instead of a more individualized approach [53] . Further studies will elucidate whether the use of muscle ultrasound may play a role in the optimization and individualization of nutrient delivery and potentially providing a tool to evaluate the effect of different nutrition regimens. As for the association between a positive cumulative fluid balance and a worse outcome, this has been known for quite a long time in the general ICU population and in septic patients [63, 64] . Unfortunately, we did not collect enough data to explain the possible reasons for a positive cumulative fluid balance in non-survivors, although this might depend on the higher severity of non-survivors (higher SAPS II score at admission, higher Ferritin and Interleukin-6) which might have caused a greater hemodynamic impact and prompted a more aggressive fluid resuscitation. As we did not measure diaphragm or limb muscle function, the precise pathophysiological meaning of the changes in size and echodensity and the potential mechanistic basis for their association with a negative outcome are unravelled and deserve further investigation. A strong correlation between increased echodensity and inflammation was confirmed in muscle biopsy studies [16, 65] . On the other hand, it is possible that changes in echodensity may depend on the accumulation fluid resuscitation-related tissue edema [16] . At variance with previous studies [18] , we found that changes in echodensity of both muscles were significantly related to a positive fluid balance. This may reflect the different patient population and the peculiar characteristics of COVID-19 patients, in whom endothelial activation and dysfunction play a key role in the pathogenesis of the disease, by altering the integrity of vessel barrier and potentially favouring fluid extravasation. As major limitation, we first recognize the use of predictive equation in the estimation of energy needs, and the lack of daily nitrogen balance calculations, hence protein deficit was used instead of the actual balance. Indeed, this approach has already been used and resulted in clinically meaningful association with patient outcomes [28] . Secondly, given the short-term of the inclusion period (first week of ICU stay), no volitional or long-term measures were assessed. Moreover, we did not record structural aspects of the muscle such as the pennation angle or fascicle length, which may be associated with the force-generating capacity of the muscle [43] . Third, measurements of diaphragm thickness can be difficult in some individuals as a poor acoustic window occurs in <5% of ICU patients [66] , and even less when the right hemidiaphragm is imaged. Moreover, it is known that adiposity might have a negative impact on the quality of ultrasound imaging [67] , and the average BMI of the population we enrolled indicated that about half of the patients can be defined as obese. However, in the present investigation, the right hemidiaphragm could be assessed by ultrasound and images were recorded and analysed in all the patients. Fourth, given the limited dimension of the diaphragm (generally around 2mm), results of the investigation of the diaphragm might be underpowered as compared to those for the rectus femoris. Finally, the observational nature and limited sample size of our study do not allow for any inference about causality to be made: the results are hypothesis-generating only, and the association a higher protein deficit, a reduced muscle mass and a higher mortality might be confounded by less severely ill patients reaching higher protein intakes. In conclusion, our data suggests that early changes in muscle size and quality may potentially be related to the outcome and be influenced by nutritional and fluid management strategies. While we wait for further, larger investigations to confirm our findings, we hypothesize that early identification of patients with or at risk for muscle wasting with muscle ultrasound may help promoting individualized nutritional interventions or earlier allocation / enhanced intensity of physical rehabilitation aiming to preserve respiratory and limb muscle size and architecture. 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