Perioperative Factors and Radiographic Severity Scores for Predicting the Duration of Mechanical Ventilation After Arterial Switch Surgery.

OBJECTIVES: Cardiac surgery on cardiopulmonary bypass (CPB) during the neonatal period can cause perioperative organ injuries. The primary aim of this study was to determine the incidence and risk factors associated with postoperative mechanical ventilation duration and acute lung injury after the arterial switch operation (ASO). The secondary aim was to examine the utility of the Brixia score for characterizing postoperative acute lung injury (ALI). DESIGN: A retrospective study. SETTING: A single-center university hospital. PARTICIPANTS: A total of 93 neonates with transposition of great arteries with intact ventricular septum (dTGA IVS) underwent ASO. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: From January 2015 to December 2022, 93 neonates with dTGA IVS were included in the study. The cohort had a median age of 4.0 (3.0-5.0) days and a mean weight of 3.3 ± 0.5 kg. About 63% of patients had ≥48 hours of postoperative mechanical ventilation after ASO. Risk factors included prematurity, post-CPB transfusion of salvaged red cells, platelets and cryoprecipitate, and postoperative fluid balance by univariate analysis. The larger transfused platelet volume was associated with the risk of ALI by multivariate analysis. The median baseline Brixia scores were 11.0 (9.0-12.0) and increased significantly in the postoperative day 1 in patients who developed moderate ALI 24 hours after admission to the intensive care unit (15.0 [13.0-16.0] v 12.0 [10.0-14.0], p = 0.046). CONCLUSIONS: Arterial switch operation results in a high incidence of ≥48-hour postoperative mechanical ventilation. Blood component transfusion is a potentially modifiable risk factor. The Brixia scores also may be used to characterize postoperative acute lung injury.

Narrative review of systemic inflammatory response mechanisms in cardiac surgery and immunomodulatory role of anesthetic agents.

Although surgical techniques and perioperative care have made significant advances, perioperative mortality in cardiac surgery remains relatively high. Single- or multiple-organ failure remains the leading cause of postoperative mortality. Systemic inflammatory response syndrome (SIRS) is a common trigger for organ injury or dysfunction in surgical patients. Cardiac surgery involves major surgical dissection, the use of cardiopulmonary bypass (CPB), and frequent blood transfusions. Ischemia-reperfusion injury and contact activation from CPB are among the major triggers for SIRS. Blood transfusion can also induce proinflammatory responses. Here, we review the immunological mechanisms of organ injury and the role of anesthetic regimens in cardiac surgery.

Volatile anesthetic isoflurane exposure facilitates Enterococcus biofilm infection.

Enterococcus faecalis (E. faecalis) is one of the major pathogenic bacteria responsible for surgical site infections. Biofilm infections are major hospital-acquired infections. Previous studies suggested that ions could regulate biofilm formation in microbes. Volatile anesthetics, frequently administered in surgical setting, target ion channels. Here, we investigated the role of ion channels/transporters and volatile anesthetics in the biofilm formation by E. faecalis MMH594 strain and its ion transporter mutants. We found that a chloride transporter mutant significantly reduced biofilm formation compared to the parental strain. Downregulation of teichoic acid biosynthesis in the chloride transporter mutant impaired biofilm matrix formation and cellular adhesion, leading to mitigated biofilm formation. Among anesthetics, isoflurane exposure enhanced biofilm formation in vitro and in vivo. The upregulation of de novo purine biosynthesis pathway by isoflurane exposure potentially enhanced biofilm formation, an essential process for DNA, RNA, and ATP synthesis. We also demonstrated that isoflurane exposure to E. faecalis increased cyclic-di-AMP and extracellular DNA production, consistent with the increased purine biosynthesis. We further showed that isoflurane enhanced the enzymatic activity of phosphoribosyl pyrophosphate synthetase (PRPP-S). With the hypothesis that isoflurane directly bound to PRPP-S, we predicted isoflurane binding site on it using rigid docking. Our study provides a better understanding of the underlying mechanisms of E. faecalis biofilm formation and highlights the potential impact of an ion transporter and volatile anesthetic on this process. These findings may lead to the development of novel strategies for preventing E. faecalis biofilm formation and improving patient outcomes in clinical settings.

Differential effects of dexmedetomidine on Gram-positive and Gram-negative bacterial killing and phagocytosis.

Dexmedetomidine is a commonly used sedative in perioperative and intensive care settings with purported immunomodulatory properties. Since its effects on immune functions against infections have not been extensively studied, we tested the effects of dexmedetomidine on Gram-positive [Staphylococcus aureus and Enterococcus faecalis] and Gram-negative bacteria [Escherichia coli], and on effector functions of human monocytes THP-1 cells against them. We evaluated phagocytosis, reactive oxygen species (ROS) formation, and CD11b activation, and performed RNA sequencing analyses. Our study revealed that dexmedetomidine improved Gram-positive but mitigated Gram-negative bacterial phagocytosis and killing in THP-1 cells. The attenuation of Toll-like receptor 4 (TLR4) signaling by dexmedetomidine was previously reported. Thus, we tested TLR4 inhibitor TAK242. Similar to dexmedetomidine, TAK242 reduced E. coli phagocytosis but enhanced CD11b activation. The reduced TLR4 response potentially increases CD11b activation and ROS generation and subsequently enhances Gram-positive bacterial killing. Conversely, dexmedetomidine may inhibit the TLR4-signaling pathway and mitigate the alternative phagocytosis pathway induced by TLR4 activation through LPS-mediated Gram-negative bacteria, resulting in worsened bacterial loads. We also examined another α2 adrenergic agonist, xylazine. Because xylazine did not affect bacterial clearance, we proposed that dexmedetomidine may have an off-target effect on bacterial killing process, potentially involving crosstalk between CD11b and TLR4. Despite its potential to attenuate inflammation, we provide a novel insight into potential risks of dexmedetomidine use during Gram-negative infections, highlighting the differential effect of dexmedetomidine on Gram-positive and Gram-negative bacteria.

Surgical site infection in pediatric spinal fusion surgery revisited: outcome and risk factors after preventive bundle implementation.

Background: Surgical site infections (SSI) contribute to significant morbidity, mortality, length of stay, and financial burden. We sought to evaluate the incidence and risk factors of surgical site infection following pediatric spinal fusion surgery in patients for whom standard perioperative antibiotic prophylaxis and preventive strategies have been implemented. Methods: We conducted a retrospective study of children aged <18 years who underwent spinal fusion surgery from January 2017 to November 2021 at a quaternary academic pediatric medical center. Univariable analysis was used to evaluate associations between potential risk factors and SSI. Results: Of 1111 patients, 752 (67.6%) were female; median age was 14.2 years. SSI occurred in 14 patients (1.3%). Infections were superficial incisional (n=2; 14.3%), deep incisional (n=9; 64.3%), and organ/space (n=3; 21.4%). Median time to SSI was 14 days (range, 8 to 45 days). Staphylococcus aureus and Escherichia coli were the most frequently-isolated bacteria. Potential risk factors for SSIs included low body weight (Odds ratio (OR) 0.96, 95% confidence interval (CI) 0.93-0.99, p=0.026), ASA classification of ≥3 (OR 24.53, 95%CI 3.20-188.22, p=0.002), neuromuscular scoliosis (OR 3.83, 95%CI 3.82-78.32, p<0.001), prolonged operative time (OR 1.56, 95%CI 1.28-1.92, p<0.001), prolonged anesthetic time (OR 1.65, 95%CI 1.35-2.00, p<0.001), administration of prophylactic antibiotic ≥60 minutes before skin incision (OR 11.52, 95%CI 2.34-56.60, p=0.003), and use of povidone-iodine alone for skin preparation (OR 5.97, 95%CI 1.27-28.06, p=0.024). Conclusion: In the context of a robust bundle for SSI prevention; low body weight, ASA classification of ≥3, neuromuscular scoliosis, prolonged operative and anesthetic times, administration of prophylactic antibiotic ≥60 minutes before skin incision, and use of povidone-iodine alone for skin preparation increased the risk of SSI. Administration of prophylactic antibiotic within 60 minutes of skin incision, strict adherence to high-risk preventive protocol, and use of CHG-alcohol could potentially reduce the rate of SSI.

Neutrophil extracellular traps formation is associated with postoperative complications in neonates and infants undergoing congenital cardiac surgery.

Pediatric patients with congenital heart diseases (CHD) often undergo surgical repair on cardiopulmonary bypass (CPB). Despite a significant medical and surgical improvement, the mortality of neonates and infants remains high. Damage-associated molecular patterns (DAMPs) are endogenous molecules released from injured/damaged tissues as danger signals. We examined 101 pediatric patients who underwent congenital cardiac surgery on CPB. The mortality rate was 4.0%, and the complication rate was 31.6%. We found that neonates/infants experienced multiple complications most, consistent with the previous knowledge. Neonates and infants in the complication group had received more transfusion intraoperatively than the non-complication arm with lower maximum amplitude (MA) on rewarming CPB thromboelastography (TEG). Despite TEG profiles were comparable at ICU admission between the two groups, the complication arm had higher postoperative chest tube output, requiring more blood transfusion. The complication group showed greater neutrophil extracellular traps (NETs) formation at the end of CPB and postoperatively. Plasma histones and high mobility group box 1 (HMGB1) levels were significantly higher in the complication arm. Both induced NETs in vitro and in vivo . As histones and HMGB1 target Toll-like receptor (TLR)2 and TLR4, their mRNA expression in neutrophils was upregulated in the complication arm. Taken together, NETs play a major role in postoperative complication in pediatric cardiac surgery and would be considered a target for intervention. Key points: Neonates and infants showed highest postoperative complications with more upregulation of inflammatory transcriptomes of neutrophils.Neonates and infants with organ dysfunction had more NETs formation with higher plasma histones and HMGB1 levels.

GltS regulates biofilm formation in methicillin-resistant Staphylococcus aureus.

Biofilm-based infection is a major healthcare burden. Methicillin-resistant Staphylococcus aureus (MRSA) is one of major organisms responsible for biofilm infection. Although biofilm is induced by a number of environmental signals, the molecule responsible for environmental sensing is not well delineated. Here we examined the role of ion transporters in biofilm formation and found that the sodium-glutamate transporter gltS played an important role in biofilm formation in MRSA. This was shown by gltS transposon mutant as well as its complementation. The lack of exogenous glutamate also enhanced biofilm formation in JE2 strain. The deficiency of exogenous glutamate intake accelerated endogenous glutamate/glutamine production, which led to the activation of the urea cycle. We also showed that urea cycle activation was critical for biofilm formation. In conclusion, we showed that gltS was a critical regulator of biofilm formation by controlling the intake of exogenous glutamate. An intervention to target glutamate intake may be a potential useful approach against biofilm.

Use of clindamycin as an alternative antibiotic prophylaxis.

Background: Clindamycin serves as an alternative surgical prophylactic antibiotic in patients with penicillin (PCN) or cephalosporin allergy labels. In the previous reports, the use of clindamycin was associated with higher incidences of surgical site infections (SSIs). We aimed to determine the characteristics of PCN or cephalosporin allergic reactions to stratify patient's risk and indicate subsequent management; leading to de-labeling of PCN or cephalosporin allergy. Methods: We conducted a prospective cohort study of patients receiving clindamycin as surgical antibiotic prophylaxis from September 2021 to March 2022. Data were collected from electronic medical records; included demographic data, antibiotic allergy labels, allergic reaction, and allergy testing. Results: Clindamycin was administered in 445 patients who underwent 451 operations. Among these patients, 53.0% (n = 236) were female with a median age of 15 years (range; 0.5-57.0 years). PCN and cephalosporin allergies were labelled in 83.8% (n = 373) and 25.6% (n = 114) patients, respectively; 11.4% (n = 51) of patients were allergic to both classes of the antibiotics. There were 191 (51.2%) and 73 (64.0%) possible hypersensitivity reactions (HSRs) in PCN and cephalosporin groups, respectively. The most common reactions were rash (PCN: n = 99, 26.5%; cephalosporin: n = 35, 30.7%), and hives (PCN: n = 71, 19.0%; cephalosporin: n = 24, 21.1%). Severe reactions included angioedema (PCN: n = 7, 1.9%; cephalosporin: n = 5, 4.4%), anaphylaxis (PCN: n = 8, 2.1%; cephalosporin: n = 7, 6.1%), bronchospasm (cephalosporin: n = 1, 0.9%), airway involvement (PCN: n = 1, 0.3%; cephalosporin: n = 1, 0.9%), serum sickness (PCN: n = 1, 0.3%), blisters (PCN: n = 1, 0.3%), and drug reaction with eosinophilia and systemic symptoms (DRESS) (PCN: n = 1, 0.3%). Low-risk history of allergy included gastrointestinal side effects (PCN: n = 9, 2.4%; cephalosporin: n = 3, 2.7%), positive family history (PCN: n = 7, 1.9%; cephalosporin: n = 1, 0.9%), and remote history of allergy (PCN: n = 2, 0.5%). There were 201 (53.9%) and 53 (46.5%) unknown reactions in PCN and cephalosporin groups, respectively. In the overall cohort, 3 patients (0.7%) were skin tested for drug allergy (PCN: n = 2, 0.5%; cephalosporin: n = 2, 1.8%). Conclusion: Clindamycin was largely administered in patients with non-severe HSRs, low-risk history or unknown reactions to PCN or cephalosporin, whom cefazolin could have been administered safely. Obtaining a detailed history of antibiotic allergy, allergy testing and/or direct oral challenge can de-label unsubstantiated PCN or cephalosporin allergy and ultimately reduce the incidence of SSIs by optimizing the rate of more effective antibiotic administration.

Propofol directly binds to and inhibits TLR7.

Sedatives/anesthetics are important medical tools to facilitate medical care and increase patients' comfort. Increasingly, there is recognition that sedatives/anesthetics can modulate immune functions. Toll-like receptors (TLRs) are major pattern recognition receptors involved in the recognition of microbial components. TLR7 recognizes single-strand RNA virus such as influenza and SARS-CoV2 viruses and initiates interferon (IFN) responses. IFN production triggered by TLR7 stimulation is a critical anti-viral response. For example, patients with TLR7 variants including loss-of- function variants were associated with severe COVID-19. Taken together, it is important to determine if sedatives/anesthetics mitigate TLR7 function. We have previously showed that TLR7-mediated activation was not affected by volatile anesthetics. However, we found that propofol attenuated TLR7 activation among intravenous sedatives in the reporter assay. TLR7 agonist R837 stimulation increased TNF-α, IL-1ß, IL-6, IL-10, and IFN-ß mRNA levels in bone marrow-derived dendritic cells, while these levels were attenuated by propofol. Our murine lung slice experiments showed that propofol attenuated IFN production. R837 increased IFN-ß expression in the lungs, and propofol attenuated IFN-ß expression in an in vivo model of R837 intranasal instillation. We also found that propofol directly bound to and hindered its association of TLR7 with MyD88. Our analysis using fropofol, propofol derivative showed that the hydroxyl group in propofol was important for propofol-TLR7 interaction.

Predictive Factors for Postoperative Intensive Care Unit Admission and Mechanical Ventilation After Cardiac Catheterization for Pediatric Pulmonary Vein Stenosis.

OBJECTIVE: To investigate the predictive factors for postoperative intensive care unit (ICU) admission and mechanical ventilation (MV) after cardiac catheterization for pediatric pulmonary vein stenosis (PVS). DESIGN: A retrospective observational study. SETTING: At a single tertiary academic pediatric medical center. PARTICIPANTS: Four hundred seventy-three pediatric patients diagnosed with PVS and who underwent cardiac catheterization from 2011 to 2021. MEASUREMENTS AND MAIN RESULTS: Univariate and multivariate analyses were performed for associated risk factors. The incidence of postoperative ICU admission was 46.5% (n = 220); among these, 93.4% (n = 206) required MV. Independent risk factors included male sex (odds ratio [OR] 3.93, p = 0.002 in ICU group; OR 2.89, p = 0.013 in MV group), low body weight (OR 0.80, p = 0.003 in ICU group; OR 0.79, p = 0.002 in MV group), preoperative oxygen supplement (OR 4.01, p = 0.002 in ICU group; OR 3.67, p = 0.003 in MV group), high PVS severity score (OR 1.15, p = 0.028 in ICU group), intraoperative hypotension requiring inotrope (OR 4.03, p = 0.011 in ICU group; OR 2.89, p = 0.035 in MV group), intraoperative red blood cell transfusion (OR 3.25, p = 0.023 in ICU group; OR 4.09, p = 0.005 in MV group), low preintervention PaO2/FIO2 ratio (OR 0.63, p = 0.009 in ICU group; OR 0.59, p = 0.002 in MV group), and high preintervention right ventricular systolic pressure (OR 1.39, p = 0.004 in ICU group; OR 1.27, p = 0.023 in MV group). CONCLUSION: The incidences of postoperative ICU admission and MV in this subgroup were relatively high. The identification of risk factors is useful in predicting and triaging the need for postoperative ICU admission and MV for the improvement of patient care.

Plasma concentrations of cefazolin in pediatric patients undergoing cardiac surgery.

Background: The guideline for antibiotic prophylaxis in pediatric cardiac surgery is currently unavailable, and the effects of cardiopulmonary bypass (CPB) may result in low plasma cefazolin concentrations and subsequent postoperative surgical site infections (SSIs). Aims: To demonstrate the calculated-unbound plasma concentrations of cefazolin during uncomplicated pediatric cardiac surgery. Settings and Design: A prospective observational study that included 18 patients

Perioperative risk factors for delayed extubation after acute type A aortic dissection surgery.

BACKGROUND: Delayed extubation after cardiac surgery is associated with high morbidity and mortality, increased intensive care unit length of stay, and healthcare cost. Acute type A aortic dissection (ATAAD) generally results in prolonged mechanical ventilation due to the complexity of surgical management and some postoperative complications. This study aimed to elucidate the perioperative risk factors for delayed extubation in patients undergoing ATAAD surgery. METHODS: A retrospective cohort study including 239 patients who were diagnosed with ATAAD and underwent emergency surgery from October 2004 to January 2018 was performed. The potential perioperative risk factors for delayed extubation were collected. This study defined delayed extubation as the time to commence extubation being greater than 48 hours. The clinical data were analyzed with univariate and multivariate analyses to identify risk factors for delayed extubation following ATAAD surgery. RESULTS: The incidence of delayed extubation was 48.5% (n=116). Multiple logistic regression analysis showed perioperative risk factors for delayed extubation included preoperative cardiac tamponade [odds ratio (OR) 3.94, 95% confidence interval (CI) 1.39-11.17, P=0.010], central arterial cannulation (ascending aorta and proximal aortic arch) for cardiopulmonary bypass (CPB) (OR 4.04, 95% CI: 1.03-15.91, P=0.046), postoperative stroke (OR 10.58, 95% CI: 2.65-42.25, P=0.001), postoperative renal dysfunction that required temporary hemodialysis (OR 6.60 95% CI: 1.97-22.11, P=0.002), and re-exploration to stop bleeding (OR 2.65, 95% CI: 1.00-6.99, P=0.050). CONCLUSIONS: Preoperative cardiac tamponade, central arterial cannulation for CPB, postoperative stroke, postoperative renal dysfunction that required temporary hemodialysis, and re-exploration to stop bleeding are perioperative risk factors for delayed extubation. Identification of the potential risk factors for delayed extubation may help optimize the perioperative management and improve postoperative outcomes of patients undergoing ATAAD surgery.

Measurement of aortic root dimensions by transesophageal echocardiography in adult patients undergoing cardiac surgery.

Background: Normal aortic root dimensions were established from studies from Western countries. As the body size is significantly associated with the aortic root dimensions, Thai populations may have smaller aortic root diameters. Aims: To elucidate the aortic root dimensions using transesophageal echocardiography (TEE) in adult patients undergoing cardiac surgery. Settings and Design: A retrospective cohort study including 150 patients aged >18 years undergoing cardiac surgery. Materials and Methods: Aortic root dimensions (annulus, sinus of Valsalva, sinotubular junction (STJ), and proximal ascending aorta) were measured using two-dimensional TEE. Patients with aortic-root related pathology were excluded. Results: Men constituted 60% of the study population; the mean age was 61.9 ± 12.6 years, and mean body surface area (BSA) was 1.7 ± 0.2 m2. The absolute dimensions for the annulus, sinus of Valsalva, STJ, and proximal ascending aorta were 22.3 ± 3.4, 32.6 ± 3.9, 26.4 ± 3.3, and 29.3 ± 3.1 mm, respectively, in men (12.9 ± 1.6, 18.8 ± 2.6, 15.2 ± 2.1, and 17.9 ± 2.7 mm, respectively, after adjusting for BSA) and 20.3 ± 2.2, 29.8 ± 3.6, 23.8 ± 2.6, and 27.1 ± 3.1 mm, respectively, in women (13.5 ± 2.0, 19.8 ± 2.3, 15.8 ± 2.5, and 17.0 ± 2.1 mm, respectively, after adjusting for BSA). The absolute aortic root diameters were significantly greater in men at all levels (P < 0.001). The BSA-adjusted diameters were similar for both sexes at the annulus (P = 0.076) and STJ (P = 0.076), except for the sinus of Valsalva (P = 0.010) and proximal ascending aorta (P = 0.006). Conclusion: This study reports reference values of aortic root dimensions by TEE. The body size should be considered when comparing the aortic root dimensions of Thai populations with the standard normal values.

Efficacy of surgical transversus abdominis plane block for postoperative pain relief following abdominal surgery in pediatric patients.

BACKGROUND: Transversus abdominis plane (TAP) block is a promising effective method for postoperative pain control after major abdominal surgery. Using a landmark technique, it is easily performed, but its popularity has decreased because of less efficacy due to inaccurate injection and the potential for intraperitoneal organ damage. Ultrasound-guided TAP block provides better results and less complications, but it requires experienced operators. Surgically administered TAP (sTAP) block is a simple technique and may cause less complications. This study was aimed to determine the efficacy of sTAP on postoperative pain control in pediatric patients following a major abdominal surgery, compared with local anesthetic infiltration and no block. METHODS: This stratified, randomized controlled trial was conducted in pediatric patients, below the age of 15 years, who underwent non-laparoscopic major abdominal surgery. Patients were allocated into three groups. The control group received no block; the LA group received 0.25% bupivacaine for local wound infiltration; and the sTAP group received 0.25% bupivacaine for TAP block performed by a surgeon before abdominal wall closure. Parameter records included the incidence of inadequate pain control, time to first analgesic, opioid requirement within 24 h, and complications of these techniques. RESULTS: Fifty-four patients were recruited. There was no significant difference in the incidence of inadequate pain control (P = 0.589). The median time to first analgesic was 380 min in the sTAP group compared with 370 and 420 min in the LA and control groups, respectively (95%CI = 193-567, 121-619, and 0-1012; P = 0.632). The median dose of total opioid requirement (calculated as fentanyl-equivalent dose) was 1.95, 2.05, and 2.04 µg·kg(-1) ·24 h(-1) in the sTAP, LA, and control groups, respectively (IQR = 0.65, 2.20; 0.59, 3.32; 0.38, 2.60; P = 0.723). No complications of sTAP block were detected. CONCLUSIONS: There was no significant advantage of the sTAP block over local infiltration or no intervention for postoperative pain control in pediatric patients undergoing non-laparoscopic major abdominal surgeries.

Plasma concentrations of bupivacaine after spinal anesthesia with single shot femoral nerve block and periarticular injection in total knee arthroplasty.

OBJECTIVE: To determine the plasma concentrations of bupivacaine and toxicity after periarticular injection (PAI) combined with spinal anesthesia and femoral nerve block (FNB). MATERIAL AND METHOD: Forty-three patients scheduled for unilateral total knee arthroplasty (TKA) were enrolled in the prospective observational study. The dose of bupivacaine for spinal anesthesia was adjusted by the attending anesthesiologist. The single-shot femoral nerve block (FNB) and periarticular injection (PAI) were performed with 20 ml of 0.5% bupivacaine and 20 ml of 0.25% bupivacaine respectively. Venous blood samples from antecubital vein were collected at 60 minutes after femoral nerve block and at the time before periarticular injection, then at 15, 30, 45, and 60 minutes afterwards. Plasma bupivacaine concentrations were analyzed, using a high performance liquid chromatography with tandem mass spectrometry. RESULTS: Ten males and 32 females, ASA I-II were included. The highest median plasma concentration was 586.22 ng/ml (min = 245.39, max = 1,614.36) at 45 minutes after periarticular injection. The maximum plasma bupivacaine concentration was 1,709.71 ng/ml at 60 minutes after periarticular injection. No clinical toxicity was encountered CONCLUSION: The plasma concentration of bupivacaine in patients performed periarticular injection with 20 ml of 0.25% bupivacaine after spinal anesthesia and single-shot femoral nerve block with 20 ml of 0.5% bupivacaine is below the plasma toxic level.