Non-targeted analytical comparison of a heated tobacco product aerosol against mainstream cigarette smoke: does heating tobacco produce an inherently different set of aerosol constituents?

Smoking-related diseases remain a significant public health concern, and heated tobacco products (HTPs) have emerged as a potential alternative to cigarettes. While several studies have confirmed that HTP aerosols contain lower levels of harmful and potentially harmful constituents (HPHCs) than cigarette smoke, less is known about constituents that are intrinsically higher in HTP aerosols. This study provides a comprehensive comparative assessment of an HTP aerosol produced with Tobacco Heating System 2.2 (THS) and comparator cigarette (CC) smoke aiming at identifying all unique or increased compounds in THS aerosol by applying a broad set of LC-MS and GC × GC-MS methods. To focus on differences due to heating versus burning tobacco, confounding factors were minimized by using the same tobacco in both test items and not adding flavorants. Of all analytical features, only 3.5%-corresponding to 31 distinctive compounds-were significantly more abundant in THS aerosol than in CC smoke. A notable subset of these compounds was identified as reaction products of glycerol. The only compound unique to THS aerosol was traced back to its presence in a non-tobacco material in the test item and not a direct product of heating tobacco. Our results demonstrate that heating a glycerol-containing tobacco substrate to the temperatures applied in THS does not introduce new compounds in the resulting aerosol compared to CC smoke which are detectable with the method portfolio applied in this study. Overall, this study contributes to a better understanding of the chemical composition of HTP aerosols and their potential impact on human health.

Treatment Practices and Outcomes in Continuous Spike and Wave during Slow Wave Sleep: A Multicenter Collaboration.

OBJECTIVES: To determine how continuous spike and wave during slow wave sleep (CSWS) is currently managed and to compare the effectiveness of current treatment strategies using a database from 11 pediatric epilepsy centers in the US. STUDY DESIGN: This retrospective study gathered information on baseline clinical characteristics, CSWS etiology, and treatment(s) in consecutive patients seen between 2014 and 2016 at 11 epilepsy referral centers. Treatments were categorized as benzodiazepines, steroids, other antiseizure medications (ASMs), or other therapies. Two measures of treatment response (clinical improvement as noted by the treating physician; and electroencephalography improvement) were compared across therapies, controlling for baseline variables. RESULTS: Eighty-one children underwent 153 treatment trials during the study period (68 trials of benzodiazepines, 25 of steroids, 45 of ASMs, 14 of other therapies). Children most frequently received benzodiazepines (62%) or ASMs (27%) as first line therapy. Treatment choice did not differ based on baseline clinical variables, nor did these variables correlate with outcome. After adjusting for baseline variables, children had a greater odds of clinical improvement with benzodiazepines (OR 3.32, 95%CI 1.57-7.04, P = .002) or steroids (OR 4.04, 95%CI 1.41-11.59, P = .01) than with ASMs and a greater odds of electroencephalography improvement after steroids (OR 3.36, 95% CI 1.09-10.33, P = .03) than after ASMs. CONCLUSIONS: Benzodiazepines and ASMs are the most frequent initial therapy prescribed for CSWS in the US. Our data suggests that ASMs are inferior to benzodiazepines and steroids and support earlier use of these therapies. Multicenter prospective studies that rigorously assess treatment protocols and outcomes are needed.

A complex matrix characterization approach, applied to cigarette smoke, that integrates multiple analytical methods and compound identification strategies for non-targeted liquid chromatography with high-resolution mass spectrometry.

RATIONALE: For the characterization of the chemical composition of complex matrices such as tobacco smoke, containing more than 6000 constituents, several analytical approaches have to be combined to increase compound coverage across the chemical space. Furthermore, the identification of unknown molecules requiring the implementation of additional confirmatory tools in the absence of reference standards, such as tandem mass spectrometry spectra comparisons and in silico prediction of mass spectra, is a major bottleneck. METHODS: We applied a combination of four chromatographic/ionization techniques (reversed-phase (RP) - heated electrospray ionization (HESI) in both positive (+) and negative (-) modes, RP - atmospheric pressure chemical ionization (APCI) in positive mode, and hydrophilic interaction liquid chromatography (HILIC) - HESI positive) using a Thermo Q Exactive™ liquid chromatography/high-resolution accurate mass spectrometry (LC/HRAM-MS) platform for the analysis of 3R4F-derived smoke. Compound identification was performed by using mass spectral libraries and in silico predicted fragments from multiple integrated databases. RESULTS: A total of 331 compounds with semi-quantitative estimates ≥100 ng per cigarette were identified, which were distributed within the known chemical space of tobacco smoke. The integration of multiple LC/HRAM-MS-based chromatographic/ionization approaches combined with complementary compound identification strategies was key for maximizing the number of amenable compounds and for strengthening the level of identification confidence. A total of 50 novel compounds were identified as being present in tobacco smoke. In the absence of reference MS2 spectra, in silico MS2 spectra prediction gave a good indication for compound class and was used as an additional confirmatory tool for our integrated non-targeted screening (NTS) approach. CONCLUSIONS: This study presents a powerful chemical characterization approach that has been successfully applied for the identification of novel compounds in cigarette smoke. We believe that this innovative approach has general applicability and a huge potential benefit for the analysis of any complex matrices.

Comprehensive chemical characterization of the aerosol generated by a heated tobacco product by untargeted screening.

A suite of untargeted methods has been applied for the characterization of aerosol from the Tobacco Heating System 2.2 (THS2.2), a heated tobacco product developed by Philip Morris Products S.A. and commercialized under the brand name IQOS®. A total of 529 chemical constituents, excluding water, glycerin, and nicotine, were present in the mainstream aerosol of THS2.2, generated by following the Health Canada intense smoking regimen, at concentrations ≥ 100 ng/item. The majority were present in the particulate phase (n = 402), representing more than 80% of the total mass determined by untargeted screening; a proportion were present in both particulate and gas-vapor phases (39 compounds). The identities for 80% of all chemical constituents (representing > 96% of the total determined mass) were confirmed by the use of authentic analytical reference materials. Despite the uncertainties that are recognized to be associated with aerosol-based untargeted approaches, the reported data remain indicative that the uncharacterized fraction of TPM generated by THS2.2 has been evaluated to the fullest practicable extent. To the best of our knowledge, this work represents the most comprehensive chemical characterization of a heated tobacco aerosol to date. Graphical abstract.

Genetic and phenotypic heterogeneity suggest therapeutic implications in SCN2A-related disorders.

Mutations in SCN2A, a gene encoding the voltage-gated sodium channel Nav1.2, have been associated with a spectrum of epilepsies and neurodevelopmental disorders. Here, we report the phenotypes of 71 patients and review 130 previously reported patients. We found that (i) encephalopathies with infantile/childhood onset epilepsies (≥3 months of age) occur almost as often as those with an early infantile onset (<3 months), and are thus more frequent than previously reported; (ii) distinct phenotypes can be seen within the late onset group, including myoclonic-atonic epilepsy (two patients), Lennox-Gastaut not emerging from West syndrome (two patients), and focal epilepsies with an electrical status epilepticus during slow sleep-like EEG pattern (six patients); and (iii) West syndrome constitutes a common phenotype with a major recurring mutation (p.Arg853Gln: two new and four previously reported children). Other known phenotypes include Ohtahara syndrome, epilepsy of infancy with migrating focal seizures, and intellectual disability or autism without epilepsy. To assess the response to antiepileptic therapy, we retrospectively reviewed the treatment regimen and the course of the epilepsy in 66 patients for which well-documented medical information was available. We find that the use of sodium channel blockers was often associated with clinically relevant seizure reduction or seizure freedom in children with early infantile epilepsies (<3 months), whereas other antiepileptic drugs were less effective. In contrast, sodium channel blockers were rarely effective in epilepsies with later onset (≥3 months) and sometimes induced seizure worsening. Regarding the genetic findings, truncating mutations were exclusively seen in patients with late onset epilepsies and lack of response to sodium channel blockers. Functional characterization of four selected missense mutations using whole cell patch-clamping in tsA201 cells-together with data from the literature-suggest that mutations associated with early infantile epilepsy result in increased sodium channel activity with gain-of-function, characterized by slowing of fast inactivation, acceleration of its recovery or increased persistent sodium current. Further, a good response to sodium channel blockers clinically was found to be associated with a relatively small gain-of-function. In contrast, mutations in patients with late-onset forms and an insufficient response to sodium channel blockers were associated with loss-of-function effects, including a depolarizing shift of voltage-dependent activation or a hyperpolarizing shift of channel availability (steady-state inactivation). Our clinical and experimental data suggest a correlation between age at disease onset, response to sodium channel blockers and the functional properties of mutations in children with SCN2A-related epilepsy.

Electrographic seizures after convulsive status epilepticus in children and young adults: a retrospective multicenter study.

OBJECTIVE: To describe the prevalence, characteristics, and predictors of electrographic seizures after convulsive status epilepticus (CSE). STUDY DESIGN: This was a multicenter retrospective study in which we describe clinical and electroencephalographic (EEG) features of children (1 month to 21 years) with CSE who underwent continuous EEG monitoring. RESULTS: Ninety-eight children (53 males) with CSE (median age of 5 years) underwent subsequent continuous EEG monitoring after CSE. Electrographic seizures (with or without clinical correlate) were identified in 32 subjects (33%). Eleven subjects (34.4%) had electrographic-only seizures, 17 subjects (53.1%) had electroclinical seizures, and 4 subjects (12.5%) had an unknown clinical correlate. Of the 32 subjects with electrographic seizures, 15 subjects (46.9%) had electrographic status epilepticus. Factors associated with the occurrence of electrographic seizures after CSE were a previous diagnosis of epilepsy (P = .029) and the presence of interictal epileptiform discharges (P < .0005). The median (p25-p75) duration of stay in the pediatric intensive care unit was longer for children with electrographic seizures than for children without electrographic seizures (9.5 [3-22.5] vs 2 [2-5] days, Wilcoxon test, Z = 3.916, P = .0001). Four children (4.1%) died before leaving the hospital, and we could not identify a relationship between death and the presence or absence of electrographic seizures. CONCLUSIONS: After CSE, one-third of children who underwent EEG monitoring experienced electrographic seizures, and among these, one-third experienced entirely electrographic-only seizures. A previous diagnosis of epilepsy and the presence of interictal epileptiform discharges were risk factors for electrographic seizures.

Computer-assisted structure identification (CASI)--an automated platform for high-throughput identification of small molecules by two-dimensional gas chromatography coupled to mass spectrometry.

Compound identification is widely recognized as a major bottleneck for modern metabolomic approaches and high-throughput nontargeted characterization of complex matrices. To tackle this challenge, an automated platform entitled computer-assisted structure identification (CASI) was designed and developed in order to accelerate and standardize the identification of compound structures. In the first step of the process, CASI automatically searches mass spectral libraries for matches using a NIST MS Search algorithm, which proposes structural candidates for experimental spectra from two-dimensional gas chromatography with time-of-flight mass spectrometry (GC × GC-TOF-MS) measurements, each with an associated match factor. Next, quantitative structure-property relationship (QSPR) models implemented in CASI predict three specific parameters to enhance the confidence for correct compound identification, which were Kovats Index (KI) for the first dimension (1D) separation, relative retention time for the second dimension separation (2DrelRT) and boiling point (BP). In order to reduce the impact of chromatographic variability on the second dimension retention time, a concept based upon hypothetical reference points from linear regressions of a deuterated n-alkanes reference system was introduced, providing a more stable relative retention time measurement. Predicted values for KI and 2DrelRT were calculated and matched with experimentally derived values. Boiling points derived from 1D separations were matched with predicted boiling points, calculated from the chemical structures of the candidates. As a last step, CASI combines the NIST MS Search match factors (NIST MF) with up to three predicted parameter matches from the QSPR models to generate a combined CASI Score representing the measure of confidence for the identification. Threshold values were applied to the CASI Scores assigned to proposed structures, which improved the accuracy for the classification of true/false positives and true/false negatives. Results for the identification of compounds have been validated, and it has been demonstrated that identification using CASI is more accurate than using NIST MS Search alone. CASI is an easily accessible web-interfaced software platform which represents an innovative, high-throughput system that allows fast and accurate identification of constituents in complex matrices, such as those requiring 2D separation techniques.

Subclinical early posttraumatic seizures detected by continuous EEG monitoring in a consecutive pediatric cohort.

PURPOSE: Traumatic brain injury (TBI) is an important cause of morbidity and mortality in children, and early posttraumatic seizures (EPTS) are a contributing factor to ongoing acute damage. Continuous video-EEG monitoring (cEEG) was utilized to assess the burden of clinical and electrographic EPTS. METHODS: Eighty-seven consecutive, unselected (mild - severe), acute TBI patients requiring pediatric intensive care unit (PICU) admission at two academic centers were monitored prospectively with cEEG per established clinical TBI protocols. Clinical and subclinical seizures and status epilepticus (SE, clinical and subclinical) were assessed for their relation to clinical risk factors and short-term outcome measures. KEY FINDINGS: Of all patients, 42.5% (37/87) had seizures. Younger age (p = 0.002) and injury mechanism (abusive head trauma - AHT, p < 0.001) were significant risk factors. Subclinical seizures occurred in 16.1% (14/87), while 6.9% (6/87) had only subclinical seizures. Risk factors for subclinical seizures included younger age (p < 0.001), AHT (p < 0.001), and intraaxial bleed (p < 0.001). SE occurred in 18.4% (16/87) with risk factors including younger age (p < 0.001), AHT (p < 0.001), and intraaxial bleed (p = 0.002). Subclinical SE was detected in 13.8% (12/87) with significant risk factors including younger age (p < 0.001), AHT (p = 0.001), and intraaxial bleed (p = 0.004). Subclinical seizures were associated with lower discharge King's Outcome Scale for Childhood Head Injury (KOSCHI) score (p = 0.002). SE and subclinical SE were associated with increased hospital length of stay (p = 0.017 and p = 0.041, respectively) and lower hospital discharge KOSCHI (p = 0.007 and p = 0.040, respectively). SIGNIFICANCE: cEEG monitoring significantly improves detection of seizures/SE and is the only way to detect subclinical seizures/SE. cEEG may be indicated after pediatric TBI, particularly in younger children, AHT cases, and those with intraaxial blood on computerized tomography (CT).

Electroencephalography monitoring in critically ill children: current practice and implications for future study design.

PURPOSE: Survey data indicate that continuous electroencephalography (EEG) (CEEG) monitoring is used with increasing frequency to identify electrographic seizures in critically ill children, but studies of current CEEG practice have not been conducted. We aimed to describe the clinical utilization of CEEG in critically ill children at tertiary care hospitals with a particular focus on variables essential for designing feasible prospective multicenter studies evaluating the impact of electrographic seizures on outcome. METHODS: Eleven North American centers retrospectively enrolled 550 consecutive critically ill children who underwent CEEG. We collected data regarding subject characteristics, CEEG indications, and CEEG findings. KEY FINDINGS: CEEG indications were encephalopathy with possible seizures in 67% of subjects, event characterization in 38% of subjects, and management of refractory status epilepticus in 11% of subjects. CEEG was initiated outside routine work hours in 47% of subjects. CEEG duration was <12 h in 16%, 12-24 h in 34%, and >24 h in 48%. Substantial variability existed among sites in CEEG indications and neurologic diagnoses, yet within each acute neurologic diagnosis category a similar proportion of subjects at each site had electrographic seizures. Electrographic seizure characteristics including distribution and duration varied across sites and neurologic diagnoses. SIGNIFICANCE: These data provide a systematic assessment of recent CEEG use in critically ill children and indicate variability in practice. The results suggest that multicenter studies are feasible if CEEG monitoring pathways can be standardized. However, the data also indicate that electrographic seizure variability must be considered when designing studies that address the impact of electrographic seizures on outcome.

Epilepsy and Electroencephalographic Abnormalities in SATB2-Associated Syndrome.

BACKGROUND: Seizures are an under-reported feature of the SATB2-associated syndrome phenotype. We describe the electroencephalographic findings and seizure semiology and treatment in a population of individuals with SATB2-associated syndrome. METHODS: We performed a retrospective review of 101 individuals with SATB2-associated syndrome who were reported to have had a previous electroencephalographic study to identify those who had at least one reported abnormal result. For completeness, a supplemental survey was distributed to the caregivers and input from the treating neurologist was obtained whenever possible. RESULTS: Forty-one subjects were identified as having at least one prior abnormal electroencephalography. Thirty-eight individuals (93%) had epileptiform discharges, 28 (74%) with central localization. Sleep stages were included as part of the electroencephalographies performed in 31 individuals (76%), and epileptiform activity was recorded during sleep in all instances (100%). Definite clinical seizures were diagnosed in 17 individuals (42%) with a mean age of onset of 3.2 years (four months to six years), and focal seizures were the most common type of seizure observed (42%). Six subjects with definite clinical seizures needed polytherapy (35%). Delayed myelination and/or abnormal white matter hyperintensities were seen on neuroimaging in 19 individuals (61%). CONCLUSIONS: Epileptiform abnormalities are commonly seen in individuals with SATB2-associated syndrome. A baseline electroencephalography that preferably includes sleep stages is recommended during the initial evaluation of all individuals with SATB2-associated syndrome, regardless of clinical suspicion of epilepsy.

A Rasmussen encephalitis, autoimmune encephalitis, and mitochondrial disease mimicker: expanding the DNM1L-associated intractable epilepsy and encephalopathy phenotype.

Dynamin-1-like protein (DNM1L) gene variants have been linked to childhood refractory epilepsy, developmental delay, encephalopathy, microcephaly, and progressive diffuse cerebral atrophy. However, only a few cases have been reported in the literature and there is still a limited amount of information about the symptomatology and pathophysiology associated with pathogenic variants of DNM1L. We report a 10-year-old girl with a one-year history of mild learning disorder and absence seizures who presented with new-onset focal status epilepticus which progressed to severe encephalopathy and asymmetric hemispheric cerebral atrophy. Differential diagnosis included mitochondrial disease, Rasmussen's encephalitis, and autoimmune encephalitis. Disease progressed from one hemisphere to the other despite anti-seizure medications, hemispherectomy, vagus nerve stimulator, ketogenic diet, and immunomodulators. Continued cerebral atrophy and refractory seizures evolved until death four years after initial presentation. Post-mortem whole-exome sequencing revealed a pathogenic DNM1L variant. This paper presents a novel case of adolescent-onset DNM1L-related intractable epilepsy and encephalopathy.

Early Posttraumatic Seizures in the Pediatric Population.

Early posttraumatic seizure is a paramount clinical issue in pediatric traumatic brain injury patients as it is a common occurrence, yet an understudied entity at present. Recent literature recognizes several posttraumatic seizure subtypes based on time of presentation and the underlying pathophysiology: impact, immediate, delayed early, and late/posttraumatic epilepsy. Appropriate classification of pediatric posttraumatic seizure subtypes can be helpful for appropriate management and prognosis. This review will focus on early posttraumatic seizures, and the subtypes of early posttraumatic seizure. Incidence, risk factors, diagnosis, seizure semiology, status epilepticus, management, risk of recurrence, and prognosis were reviewed. The integration of continuous electroencephalographic (EEG) monitoring into pediatric traumatic brain injury management may hold the key to better characterizing and understanding pediatric early posttraumatic seizures. Topics for future research pertaining to pediatric early posttraumatic seizure are identified.

Development and validation of a seizure prediction model in critically ill children.

PURPOSE: Electrographic seizures are common in encephalopathic critically ill children, but identification requires continuous EEG monitoring (CEEG). Development of a seizure prediction model would enable more efficient use of limited CEEG resources. We aimed to develop and validate a seizure prediction model for use among encephalopathic critically ill children. METHOD: We developed a seizure prediction model using a retrospectively acquired multi-center database of children with acute encephalopathy without an epilepsy diagnosis, who underwent clinically indicated CEEG. We performed model validation using a separate prospectively acquired single center database. Predictor variables were chosen to be readily available to clinicians prior to the onset of CEEG and included: age, etiology category, clinical seizures prior to CEEG, initial EEG background category, and inter-ictal discharge category. RESULTS: The model has fair to good discrimination ability and overall performance. At the optimal cut-off point in the validation dataset, the model has a sensitivity of 59% and a specificity of 81%. Varied cut-off points could be chosen to optimize sensitivity or specificity depending on available CEEG resources. CONCLUSION: Despite inherent variability between centers, a model developed using multi-center CEEG data and few readily available variables could guide the use of limited CEEG resources when applied at a single center. Depending on CEEG resources, centers could choose lower cut-off points to maximize identification of all patients with seizures (but with more patients monitored) or higher cut-off points to reduce resource utilization by reducing monitoring of lower risk patients (but with failure to identify some patients with seizures).

Electrographic seizures in pediatric ICU patients: cohort study of risk factors and mortality.

OBJECTIVES: We aimed to determine the incidence of electrographic seizures in children in the pediatric intensive care unit who underwent EEG monitoring, risk factors for electrographic seizures, and whether electrographic seizures were associated with increased odds of mortality. METHODS: Eleven sites in North America retrospectively reviewed a total of 550 consecutive children in pediatric intensive care units who underwent EEG monitoring. We collected data on demographics, diagnoses, clinical seizures, mental status at EEG onset, EEG background, interictal epileptiform discharges, electrographic seizures, intensive care unit length of stay, and in-hospital mortality. RESULTS: Electrographic seizures occurred in 162 of 550 subjects (30%), of which 61 subjects (38%) had electrographic status epilepticus. Electrographic seizures were exclusively subclinical in 59 of 162 subjects (36%). A multivariable logistic regression model showed that independent risk factors for electrographic seizures included younger age, clinical seizures prior to EEG monitoring, an abnormal initial EEG background, interictal epileptiform discharges, and a diagnosis of epilepsy. Subjects with electrographic status epilepticus had greater odds of in-hospital death, even after adjusting for EEG background and neurologic diagnosis category. CONCLUSIONS: Electrographic seizures are common among children in the pediatric intensive care unit, particularly those with specific risk factors. Electrographic status epilepticus occurs in more than one-third of children with electrographic seizures and is associated with higher in-hospital mortality.

Unrecognized secondary causes of hypertension in patients with hypertensive urgency/emergency: prevalence and co-prevalence.

BACKGROUND: Hypertensive urgency/emergency occurs frequently, yet no prospective data on common secondary causes, including sleep apnea (SA), renal artery stenosis (RAS), and hyperaldosteronism, are available. METHODS: Patients presenting to the emergency room for over 1 year with systolic blood pressure > or =180 mmHg and/or diastolic blood pressure > or =100 mmHg and typical symptoms were included. RAS was diagnosed by direct duplex/Doppler ultrasound of the renal artery, resistance index, and imaging. The aldosterone/renin ratio (ARR) was determined from morning blood samples taken with the patients supine after > or =2 h of rest. A positive ARR (>50) was followed by saline infusion to exclude primary hyperaldosteronism. SA was evaluated by nasal breathing flow screening; when positive [apnea/hypopnea index (AHI) >5/h], complete polysomnography was performed. RESULTS: Of 161 patients (age, 66.0 +/- 13.1 years; BMI, 28.6 +/- 5.1 kg), 131 had previously identified hypertension (duration, 12.7 +/- 11.5 years; 1.9 +/- 1.5 antihypertensive medications). SA was found in 114 (70.8%) patients [18% mild (AHI: 5-15/h), 26.8% moderate (15.1-30/h), and 24.2% severe (>30/h)]. Aldosterone levels exceeded 160 pg/ml in 22 of 23 patients with hyperaldosteronism; 4 had primary and 12 had secondary hyperaldosteronism. Thirteen (8.1%) patients had RAS. Three secondary causes were found in 1 patient (0.6%), > or =2 in 25 (15.5%), and > or =1 in 124 patients (77.0%). Of 150 detected secondary causes, only 5 were recognized previously. CONCLUSIONS: Secondary causes of hypertension are common and predominantly unrecognized in patients with hypertensive urgency/emergency. Co-prevalence of secondary causes occurs in about 15% and should be considered before therapeutic intervention.