Relationship between critical care nutrition and post-intensive care syndrome in surviving ventilated patients with COVID-19: a multicenter prospective observational study.

The impact of nutrition therapy in the acute phase on post-intensive care syndrome (PICS) remains unclear. We conducted a multicenter prospective study on adult patients with COVID-19 who required mechanical ventilation for more than three days. The questionnaire was mailed after discharge. Physical PICS, defined as less than 90 points on the Barthel index (BI), was assigned as the primary outcome. We examined the types of nutrition therapy in the first week that affected PICS components. 269 eligible patients were evaluated 10 months after discharge. Supplemental parenteral nutrition (SPN) >400 kcal/day correlated with a lower occurrence of physical PICS (10% vs 21.92%, p = 0.042), whereas the amounts of energy and protein provided, early enteral nutrition, and a gradual increase in nutrition delivery did not, and none correlated with cognitive or mental PICS. A multivariable regression analysis revealed that SPN had an independent impact on physical PICS (odds ratio 0.33, 95% CI 0.12-0.92, p = 0.034), even after adjustments for age, sex, body mass index and severity. Protein provision ≥1.2 g/kg/day was associated with a lower occurrence of physical PICS (odds ratio 0.42, 95% CI 0.16-1.08, p = 0.071). In conclusion, SPN in the acute phase had a positive impact on physical PICS for ventilated patients with COVID-19.

Relationship between the Rod complex and peptidoglycan structure in Escherichia coli.

Peptidoglycan for elongation in Escherichia coli is synthesized by the Rod complex, which includes RodZ. Although various mutant strains of the Rod complex have been isolated, the relationship between the activity of the Rod complex and the overall physical and chemical structures of the peptidoglycan have not been reported. We constructed a RodZ mutant, termed RMR, and analyzed the growth rate, morphology, and other characteristics of cells producing the Rod complexes containing RMR. The growth and morphology of RMR cells were abnormal, and we isolated suppressor mutants from RMR cells. Most of the suppressor mutations were found in components of the Rod complex, suggesting that these suppressor mutations increase the integrity and/or the activity of the Rod complex. We purified peptidoglycan from wild-type, RMR, and suppressor mutant cells and observed their structures in detail. We found that the peptidoglycan purified from RMR cells had many large holes and different compositions of muropeptides from those of WT cells. The Rod complex may be a determinant not only for the whole shape of peptidoglycan but also for its highly dense structure to support the mechanical strength of the cell wall.

A critical role of the periplasm in copper homeostasis in Gram-negative bacteria.

Copper is essential for life, but is toxic in excess. Copper homeostasis is achieved in the cytoplasm and the periplasm as a unique feature of Gram-negative bacteria. Especially, it has become clear the role of the periplasm and periplasmic proteins regarding whole-cell copper homeostasis. Here, we addressed the role of the periplasm and periplasmic proteins in copper homeostasis using a Systems Biology approach integrating experiments with models. Our analysis shows that most of the copper-bound molecules localize in the periplasm but not cytoplasm, suggesting that Escherichia coli utilizes the periplasm to sense the copper concentration in the medium and sequester copper ions. In particular, a periplasmic multi-copper oxidase CueO and copper-responsive transcriptional factor CusS contribute both to protection against Cu(I) toxicity and to incorporating copper into the periplasmic components/proteins. We propose that Gram-negative bacteria have evolved mechanisms to sense and store copper in the periplasm to expand their living niches.

Machine learning algorithms for predicting days of high incidence for out-of-hospital cardiac arrest.

Predicting out-of-hospital cardiac arrest (OHCA) events might improve outcomes of OHCA patients. We hypothesized that machine learning algorithms using meteorological information would predict OHCA incidences. We used the Japanese population-based repository database of OHCA and weather information. The Tokyo data (2005-2012) was used as the training cohort and datasets of the top six populated prefectures (2013-2015) as the test. Eight various algorithms were evaluated to predict the high-incidence OHCA days, defined as the daily events exceeding 75% tile of our dataset, using meteorological and chronological values: temperature, humidity, air pressure, months, days, national holidays, the day before the holidays, the day after the holidays, and New Year's holidays. Additionally, we evaluated the contribution of each feature by Shapley Additive exPlanations (SHAP) values. The training cohort included 96,597 OHCA patients. The eXtreme Gradient Boosting (XGBoost) had the highest area under the receiver operating curve (AUROC) of 0.906 (95% confidence interval; 0.868-0.944). In the test cohorts, the XGBoost algorithms also had high AUROC (0.862-0.923). The SHAP values indicated that the "mean temperature on the previous day" impacted the most on the model. Algorithms using machine learning with meteorological and chronological information could predict OHCA events accurately.

Cardiovascular autonomic dysfunction induced by mechanical insufflation-exsufflation in Guillain-Barré syndrome.

Mechanical insufflation-exsufflation (MI-E) is an effective airway clearance device for impaired cough associated with respiratory muscle weakness caused by neuromuscular disease. Its complications on the respiratory system, such as pneumothorax, are well-recognized, but the association of the autonomic nervous system dysfunction with MI-E has never been reported. We herein describe two cases of Guillain-Barré syndrome with cardiovascular autonomic dysfunction during MI-E: a 22-year-old man who developed transient asystole and an 83-year-old man who presented with prominent fluctuation of blood pressure. These episodes occurred during the use of MI-E with abnormal cardiac autonomic testing, such as heart rate variability in both patients. While Guillain-Barré syndrome itself may cause cardiac autonomic dysfunction, MI-E possibly caused or enhanced the autonomic dysfunction by an alternation of thoracic cavity pressure. The possibility of MI-E-related cardiovascular complications should be recognized, and its appropriate monitoring and management are necessary, particularly when used for Guillain-Barré syndrome patients.

Genetic analysis of Bacillus subtilis stable L-forms obtained via long-term cultivation.

Various bacteria can change to a spherical cell-wall-deficient state, called L-from, in the presence of antibiotics that inhibit cell wall synthesis. L-forms are classified into two types: unstable and stable L-forms. Unstable L-forms revert to a normal walled state in the absence of antibiotics, while stable L-forms remain in their wall-deficient state. The conversion from unstable to stable L-forms has been often observed during long-term cultivation. However, the genetic cause for this conversion is not yet fully understood. Here, we obtained stable Bacillus subtilis L-form strains from unstable L-form strains via three independent long-term culturing experiments. The whole genome sequencing of the long-cultured strains identified many mutations, and some mutations were commonly found in all three long-cultured strains. The knockout strain of one of the commonly mutated genes, tagF, in the ancestral strain lost the ability to revert to walled state (rod shape), supporting that eliminating the function of tagF gene is one of the possible methods to convert unstable L forms to a stable state.

Prevalence and Risk Factor Analysis of Post-Intensive Care Syndrome in Patients with COVID-19 Requiring Mechanical Ventilation: A Multicenter Prospective Observational Study.

INTRODUCTION: Post-intensive care syndrome (PICS) is an emerging problem in critically ill patients and the prevalence and risk factors are unclear in patients with severe coronavirus disease 2019 (COVID-19). This multicenter prospective observational study aimed to investigate the prevalence and risk factors of PICS in ventilated patients with COVID-19 after ICU discharge. METHODS: Questionnaires were administered twice in surviving patients with COVID-19 who had required mechanical ventilation, concerning Barthel Index, Short-Memory Questionnaire, and Hospital Anxiety and Depression Scale scores. The risk factors for PICS were examined using a multivariate logistic regression analysis. RESULTS: The first and second PICS surveys were obtained at 5.5 and 13.5 months (mean) after ICU discharge, with 251 and 209 patients completing the questionnaires and with a prevalence of PICS of 58.6% and 60.8%, respectively, along with the highest percentages of cognitive impairment. Delirium (with an odds ratio of (OR) 2.34, 95% CI 1.1-4.9, and p = 0.03) and the duration of mechanical ventilation (with an OR of 1.29, 95% CI 1.05-1.58, and p = 0.02) were independently identified as the risk factors for PICS in the first PICS survey. CONCLUSION: Approximately 60% of the ventilated patients with COVID-19 experienced persistent PICS, especially delirium, and required longer mechanical ventilation.

Identification of Attenuators of Transcriptional Termination: Implications for RNA Regulation in Escherichia coli.

The regulatory function of many bacterial small RNAs (sRNAs) requires the binding of the RNA chaperone Hfq to the 3' portion of the sRNA intrinsic terminator, and therefore sRNA signaling might be regulated by modulating its terminator. Here, using a multicopy screen developed with the terminator of sRNA SgrS, we identified an sRNA gene (cyaR) and three protein-coding genes (cspD, ygjH, and rof) that attenuate SgrS termination in Escherichia coli. Analyses of CyaR and YgjH, a putative tRNA binding protein, suggested that the CyaR activity was indirect and the effect of YgjH was moderate. Overproduction of the protein attenuators CspD and Rof resulted in more frequent readthrough at terminators of SgrS and two other sRNAs, and regulation by SgrS of target mRNAs was reduced. The effect of Rof, a known inhibitor of Rho, was mimicked by bicyclomycin or by a rho mutant, suggesting an unexpected role for Rho in sRNA termination. CspD, a member of the cold shock protein family, bound both terminated and readthrough transcripts, stabilizing them and attenuating termination. By RNA sequencing analysis of the CspD overexpression strain, we found global effects of CspD on gene expression across some termination sites. We further demonstrated effects of endogenous CspD under slow growth conditions where cspD is highly expressed. These findings provided evidence of changes in the efficiency of intrinsic termination, confirming this as an additional layer of the regulation of sRNA signaling. IMPORTANCE Growing evidence suggests that the modulation of intrinsic termination and readthrough of transcription is more widespread than previously appreciated. For small RNAs, proper termination plays a critical role in their regulatory function. Here, we present a multicopy screen approach to identify factors that attenuate small RNA termination and therefore abrogate signaling dependent on the small RNA. This study highlights a new aspect of regulation of small RNA signaling as well as the modulation of intrinsic termination.

Bioenergetic Balance of Continuous Venovenous Hemofiltration, a Retrospective Analysis.

(1) Background: Nutrition therapy guided by indirect calorimetry (IC) is the gold standard and is associated with lower morbidity and mortality in critically ill patients. When performing IC during continuous venovenous hemofiltration (CVVH), the measured VCO2 should be corrected for the exchanged CO2 to calculate the 'true' Resting Energy Expenditure (REE). After the determination of the true REE, the caloric prescription should be adapted to the removal and addition of non-intentional calories due to citrate, glucose, and lactate in dialysis fluids to avoid over- and underfeeding. We aimed to evaluate this bioenergetic balance during CVVH and how nutrition therapy should be adapted. (2) Methods: This post hoc analysis evaluated citrate, glucose, and lactate exchange. Bioenergetic balances were calculated based on these values during three different CVVH settings: low dose with citrate, high dose with citrate, and low dose without citrate. The caloric load of these non-intentional calories during a CVVH-run was compared to the true REE. (3) Results: We included 19 CVVH-runs. The bioenergetic balance during the low dose with citrate was 498 ± 110 kcal/day (range 339 to 681 kcal/day) or 26 ± 9% (range 14 to 42%) of the true REE. During the high dose with citrate, it was 262 ± 222 kcal/day (range 56 to 262 kcal/day) or 17 ± 11% (range 7 to 32%) of the true REE. During the low dose without citrate, the bioenergetic balance was -189 ± 77 kcal/day (range -298 to -92 kcal/day) or -13 ± 8% (range -28 to -5%) of the true REE. (4) Conclusions: Different CVVH settings resulted in different bioenergetic balances ranging from -28% up to +42% of the true REE depending on the CVVH fluids chosen. When formulating a caloric prescription during CVVH, an individual approach considering the impact of these non-intentional calories is warranted.

Constitutive expression of the global regulator AbrB restores the growth defect of a genome-reduced Bacillus subtilis strain and improves its metabolite production.

Partial bacterial genome reduction by genome engineering can improve the productivity of various metabolites, possibly via deletion of non-essential genome regions involved in undesirable metabolic pathways competing with pathways for the desired end products. However, such reduction may cause growth defects. Genome reduction of Bacillus subtilis MGB874 increases the productivity of cellulases and proteases but reduces their growth rate. Here, we show that this growth defect could be restored by silencing redundant or less important genes affecting exponential growth by manipulating the global transcription factor AbrB. Comparative transcriptome analysis revealed that AbrB-regulated genes were upregulated and those involved in central metabolic pathway and synthetic pathways of amino acids and purine/pyrimidine nucleotides were downregulated in MGB874 compared with the wild-type strain, which we speculated were the cause of the growth defects. By constitutively expressing high levels of AbrB, AbrB regulon genes were repressed, while glycolytic flux increased, thereby restoring the growth rate to wild-type levels. This manipulation also enhanced the productivity of metabolites including γ-polyglutamic acid. This study provides the first evidence that undesired features induced by genome reduction can be relieved, at least partly, by manipulating a global transcription regulation system. A similar strategy could be applied to other genome engineering-based challenges aiming toward efficient material production in bacteria.

Relation between nutrition therapy in the acute phase and outcomes of ventilated patients with COVID-19 infection: a multicenter prospective observational study.

BACKGROUND: Optimal nutrition therapy has not yet been established for the acute phase of severe coronavirus disease 2019 (COVID-19) infection. OBJECTIVES: We aimed to examine the effects of nutrition delivery in the acute phase on mortality and the long-term outcomes of post-intensive care syndrome (PICS). METHODS: A multicenter prospective study was conducted on adult patients with COVID-19 infection requiring mechanical ventilation during an intensive care unit (ICU) stay. Daily total energy (kcal/kg) and protein (g/kg) deliveries in the first week of the ICU stay were calculated. The questionnaire for PICS evaluation was mailed within a median of 6 mo after hospital discharge. The primary outcome was in-hospital mortality, and secondary outcomes were the PICS components of physical impairment, cognitive dysfunction, and mental illness. RESULTS: Among 414 eligible patients, 297 who received mechanical ventilation for 7 d or longer were examined. PICS was evaluated in 175 patients among them. High protein delivery on days 4-7 correlated with a low in-hospital mortality rate. In contrast, high protein delivery on days 1-3 correlated with physical impairment. A multivariate logistic regression analysis adjusted for age, sex, BMI, and severity revealed that average energy and protein deliveries on days 4-7 correlated with decreased in-hospital mortality (OR: 0.94; 95% CI: 0.89, 0.99; P = 0.013 and OR: 0.40; 95% CI: 0.17, 0.93; P = 0.031, respectively). Nutrition delivery did not correlate with PICS outcomes after adjustments. In the multivariate regression using a restricted cubic spline model, in-hospital mortality monotonically decreased with increases in average nutrition delivery on days 4-7. CONCLUSIONS: In patents with COVID-19 on mechanical ventilation for ≥7 d, nutrition delivery in the late period of the acute phase was monotonically associated with a decrease in in-hospital mortality. Adequate protein delivery is needed on days 4-7.This trial was registered at https://www.umin.ac.jp as UMIN000041276.

Early Elevation of Cell-Free DNA After Acute Mesenteric Ischemia in Rats.

BACKGROUND: Acute mesenteric ischemia (AMI) is challenging to diagnose in the early phase. We tested the hypothesis that blood levels of cell-free DNA would increase early after AMI. In addition, proteome analysis was conducted as an exploratory analysis to identify other potential diagnostic biomarkers. METHODS: Mesenteric ischemia, abdominal sepsis, and sham model were compared in Sprague-Dawley rats. The abdominal sepsis model was induced by cecum puncture and mesenteric ischemia model by ligation of the superior mesenteric artery. Blood levels of cell-free DNA were measured 2 h and 6 h after wound closure. Shotgun proteome analysis was performed using plasma samples obtained at the 2 h timepoint; quantitative analysis was conducted for proteins detected exclusively in the AMI models. RESULTS: Blood cell-free DNA levels at 2 h after wound closure were significantly higher in the AMI model than in the sham and the abdominal sepsis models (P < 0.05). Cell-free DNA was positively correlated with the pathologic ischemia severity score (correlation coefficient 0.793-0.834, P < 0.001). Derivative proteome analysis in blood at 2-h time point revealed higher intensity of paraoxonase-1 in the AMI models than in the abdominal sepsis models; the significantly high blood paraoxonase-1 levels in the AMI models were confirmed in a separate quantitative analysis (P = 0.015). CONCLUSIONS: Cell-free DNA was demonstrated to be a promising biomarker for the early diagnosis of mesenteric ischemia in a rat model of AMI. Paraoxonase-1 may also play a role in the differential diagnosis of mesenteric ischemia from abdominal sepsis. The current results warrant further investigation in human studies.

Whole-Genome Analysis Reveals That the Nucleoid Protein IHF Predominantly Binds to the Replication Origin oriC Specifically at the Time of Initiation.

The structure and function of bacterial chromosomes are dynamically regulated by a wide variety of nucleoid-associated proteins (NAPs) and DNA superstructures, such as DNA supercoiling. In Escherichia coli, integration host factor (IHF), a NAP, binds to specific transcription promoters and regulatory DNA elements of DNA replication such as the replication origin oriC: binding to these elements depends on the cell cycle but underlying mechanisms are unknown. In this study, we combined GeF-seq (genome footprinting with high-throughput sequencing) with synchronization of the E. coli cell cycle to determine the genome-wide, cell cycle-dependent binding of IHF with base-pair resolution. The GeF-seq results in this study were qualified enough to analyze genomic IHF binding sites (e.g., oriC and the transcriptional promoters of ilvG and osmY) except some of the known sites. Unexpectedly, we found that before replication initiation, oriC was a predominant site for stable IHF binding, whereas all other loci exhibited reduced IHF binding. To reveal the specific mechanism of stable oriC-IHF binding, we inserted a truncated oriC sequence in the terC (replication terminus) locus of the genome. Before replication initiation, stable IHF binding was detected even at this additional oriC site, dependent on the specific DnaA-binding sequence DnaA box R1 within the site. DnaA oligomers formed on oriC might protect the oriC-IHF complex from IHF dissociation. After replication initiation, IHF rapidly dissociated from oriC, and IHF binding to other sites was sustained or stimulated. In addition, we identified a novel locus associated with cell cycle-dependent IHF binding. These findings provide mechanistic insight into IHF binding and dissociation in the genome.

Ultra-deep sequencing reveals dramatic alteration of organellar genomes in Physcomitrella patens due to biased asymmetric recombination.

Destabilization of organelle genomes causes organelle dysfunction that appears as abnormal growth in plants and diseases in human. In plants, loss of the bacterial-type homologous recombination repair (HRR) factors RECA and RECG induces organelle genome instability. In this study, we show the landscape of organelle genome instability in Physcomitrella patens HRR knockout mutants by deep sequencing in combination with informatics approaches. Genome-wide maps of rearrangement positions in the organelle genomes, which exhibited prominent mutant-specific patterns, were highly biased in terms of direction and location and often associated with dramatic variation in read depth. The rearrangements were location-dependent and mostly derived from the asymmetric products of microhomology-mediated recombination. Our results provide an overall picture of organelle-specific gross genomic rearrangements in the HRR mutants, and suggest that chloroplasts and mitochondria share common mechanisms for replication-related rearrangements.

Cell Lysis Directed by SulA in Response to DNA Damage in Escherichia coli.

The SOS response is induced upon DNA damage and the inhibition of Z ring formation by the product of the sulA gene, which is one of the LexA-regulated genes, allows time for repair of damaged DNA. On the other hand, severely DNA-damaged cells are eliminated from cell populations. Overexpression of sulA leads to cell lysis, suggesting SulA eliminates cells with unrepaired damaged DNA. Transcriptome analysis revealed that overexpression of sulA leads to up-regulation of numerous genes, including soxS. Deletion of soxS markedly reduced the extent of cell lysis by sulA overexpression and soxS overexpression alone led to cell lysis. Further experiments on the SoxS regulon suggested that LpxC is a main player downstream from SoxS. These findings suggested the SulA-dependent cell lysis (SDCL) cascade as follows: SulA→SoxS→LpxC. Other tests showed that the SDCL cascade pathway does not overlap with the apoptosis-like and mazEF cell death pathways.

Impact of Enteral Nutrition Within 24 Hours Versus Between 24 and 48 Hours in Patients With Severe Acute Pancreatitis: A Multicenter Retrospective Study.

OBJECTIVES: In patients with severe acute pancreatitis (SAP), early enteral nutrition (EN) is recommended by major clinical practice guidelines, but the exact timing for the initiation of EN is unknown. METHODS: We conducted a post hoc analysis of the database for a multicenter (44 institutions) retrospective study of patients with SAP in Japan. The patients were classified into 3 groups according to the timing of EN initiation after the diagnosis of SAP: within 24 hours, between 24 and 48 hours, and more than 48 hours. The primary outcome was in-hospital mortality. RESULTS: Of the 1094 study patients, 176, 120, and 798 patients started EN within 24 hours, between 24 and 48 hours, and more than 48 hours after SAP diagnosis, respectively. On multivariable analysis, hospital mortality was significantly better with EN within 48 hours than with more than 48 hours (adjusted odds ratio, 0.49; 95% confidence interval, 0.29-0.83; P < 0.001) but did not significantly differ between the groups with EN starting within 24 hours and between 24 and 48 hours (P = 0.29). CONCLUSIONS: Enteral nutrition within 24 hours may not confer any additional benefit on clinical outcomes compared with EN between 24 and 48 hours.

Direct Observation of Conversion From Walled Cells to Wall-Deficient L-Form and Vice Versa in Escherichia coli Indicates the Essentiality of the Outer Membrane for Proliferation of L-Form Cells.

Gram-negative bacteria such as Escherichia coli are surrounded by an outer membrane, which encloses a peptidoglycan layer. Even if thinner than in many Gram-positive bacteria, the peptidoglycan in E. coli allows cells to withstand turgor pressure in hypotonic medium. In hypertonic medium, E. coli treated with a cell wall synthesis inhibitor such as penicillin G form wall-deficient cells. These so-called L-form cells grow well under anaerobic conditions (i.e., in the absence of oxidative stress), becoming deformed and dividing as L-form. Upon removal of the inhibitor, they return to the walled rod-shaped state. Recently, the outer membrane was reported to provide rigidity to Gram-negative bacteria and to strengthen wall-deficient cells. However, it remains unclear why L-form cells need the outer membrane for growth. Using a microfluidic system, we found that, upon treatment with the outer membrane-disrupting drugs polymyxin B and polymyxin B nonapeptide or with the outer membrane synthesis inhibitor CHIR-090, the cells lysed during cell deformation and division, indicating that the outer membrane was important even in hypertonic medium. L-form cells could return to rod-shaped when trapped in a narrow space, but not in a wide space, likely due to insufficient physical force. Outer membrane rigidity could be compromised by lack of outer membrane proteins; Lpp, OmpA, or Pal. Deletion of lpp caused cells to lyse during cell deformation and cell division. In contrast, ompA and pal mutants could be deformed and return to small oval cells even when less physical force was exerted. These results strongly suggest that wall-deficient E. coli cells require a rigid outer membrane to survive, but not too rigid to prevent them from changing cell shape.

Mechanical Insufflation-exsufflation for the Prevention of Ventilator-associated Pneumonia in Intensive Care Units: A Retrospective Cohort Study.

AIM: Ventilator-associated pneumonia (VAP) is the most common intensive care unit (ICU)-acquired infection. The current study aimed to assess the efficacy of mechanical insufflation-exsufflation (MI-E) in preventing VAP in critically ill patients. MATERIALS AND METHODS: This retrospective cohort study was conducted at the ICU of Chiba University Hospital between January 2014 and September 2017. The inclusion criteria were patients who required invasive mechanical ventilation ≥48 hours and those who underwent rehabilitation, including chest physical therapy (CPT). In 2015, the study institution started the use of MI-E in patients with impaired cough reflex. From January to December 2014, patients undergoing CPT were classified under the historical control group, and those who received treatment using MI-E from January 2015 to September 2017 were included in the intervention group. The patients received treatment using MI-E via the endotracheal or tracheostomy tube, with insufflation-exsufflation pressure of 15-40 cm H2O. The treatment frequency was one to three sessions daily, and a physical therapist who is experienced in using MI-E facilitated the treatment. RESULTS: From January 2015 to September 2017, 11 patients received treatment using MI-E. Of the 169 patients screened in 2014, 19 underwent CPT. The incidence of VAP was significantly different between the CPT and MI-E groups (84.2% [16/19] vs 26.4% [3/11], p = 0.011). After adjusting for covariates, a multivariate logistic regression analysis was performed, and results showed that the covariates were not associated with the incidence of VAP. CONCLUSION: This retrospective cohort study suggests that the use of MI-E in critically ill patients is independently associated with a reduced incidence of VAP. CLINICAL SIGNIFICANCE: Assessing the efficacy of MI-E to prevent VAP. HOW TO CITE THIS ARTICLE: Kuroiwa R, Tateishi Y, Oshima T, Inagaki T, Furukawa S, Takemura R, et al. Mechanical Insufflation-exsufflation for the Prevention of Ventilator-associated Pneumonia in Intensive Care Units: A Retrospective Cohort Study. Indian J Crit Care Med 2021;25(1):62-66.