Francesco Mari

Interictal scalp fast oscillations as a marker of the seizure onset zone

Objective: This study aims to identify if oscillations at frequencies higher than the traditional EEG can be recorded on the scalp EEG of patients with focal epilepsy and to analyze the association of these oscillations with interictal discharges and the seizure onset zone (SOZ). Methods: The scalp EEG of 15 patients with focal epilepsy was studied. We analyzed the rates of gamma (40–80 Hz) and ripple (>80 Hz) oscillations, their co-occurrence with spikes, the number of channels with fast oscillations inside and outside the SOZ, and the specificity, sensitivity, and accuracy of gamma, ripples, and spikes to determine the SOZ. Results: Gamma and ripples frequently co-occurred with spikes (77.5% and 63% of cases). For all events, the proportion of channels with events was consistently higher inside than outside the SOZ: spikes (100% vs 70%), gamma (82% vs 33%), and ripples (48% vs 11%); p < 0.0001. The mean rates (events/min) were higher inside than outside the SOZ: spikes (2.64 ± 1.70 vs 0.69 ± 0.26, p = 0.02), gamma (0.77 ± 0.71 vs 0.20 ± 0.25, p = 0.02), and ripples (0.08 ± 0.12 vs 0.04 ± 0.09, p = 0.04). The sensitivity to identify the SOZ was spikes 100%, gamma 82%, and ripples 48%; the specificity was spikes 30%, gamma 68%, and ripples 89%; and the accuracy was spikes 43%, gamma 70%, and ripples 81%. Conclusion: The rates and the proportion of channels with gamma and ripple fast oscillations are higher inside the SOZ, indicating that they can be used as interictal scalp EEG markers for the SOZ. These fast oscillations are less sensitive but much more specific and accurate than spikes to delineate the SOZ.

Cocaine and heroin in waste water plants: a 1-year study in the city of Florence, Italy

The diffusion and trends in use of each substance is a basic information in policy planning of strategies aiming at deterrence of drug abuse or in the organization of the fight against drug trafficking. The actual diffusion of illicit drugs in a population is hardly measurable, but, among the various measures available, the analysis of waste water plants represents one of the most reliable source of data. We analyzed waste water in order to monitor illicit drug use by local population. We investigated the use of cocaine and heroin in the city of Florence, Italy, over a 1-year (July 2006-June 2007) period using state-of-the-art measuring techniques from waste water samples. Cocaine, benzoylecgonine, and morphine were determined in water samples by gas chromatography-mass spectrometer, and the amount of illicit substance was estimated. Data indicate for cocaine a bimodal distribution (December and March), while heroin showed a main peak in April. The heroin-to-cocaine use ratio in terms of estimated doses per month ranged from 0.11 to 0.76, representing new evidence of wider distribution of cocaine than heroin in Florence. Waste water analysis can become a valuable tool in monitoring use of illicit drugs over time. In particular, it can highlight changes in the magnitude and relative use of illicit drug at a population level thereby becoming useful to develop strategies against drug trafficking and abuse. If routinely performed, it can be part of Epidemiologic Surveillance Programmes on drug abuse.

Determination of aminorex in human urine samples by GC–MS after use of levamisole

The Drug Enforcement Administration (DEA) reports that as of October 2010, 79% of all cocaine seized in the United States contained levamisole. The equine conversion of levamisole to aminorex has been demonstrated. However, the metabolic fate of levamisole in humans is unknown. Nevertheless, as aminorex is amphetamine-like and hallucinogenic, it may be used as an adulterant to increase the effects of cocaine. We report here the results of in vivo studies demonstrating for the first time that not only equine, but also canine and human metabolism all result in aminorex formation. Levamisole and aminorex were extracted from real urine samples by liquid-liquid extraction and identified and quantified by GC-MS (identification by 3 ions per substance, LLOQ at 0.15ng/ml for both).

Continuous High Frequency Activity: A peculiar SEEG pattern related to specific brain regions

OBJECTIVE While visually marking the high frequency oscillations in the stereo-EEG of epileptic patients, we observed a continuous/semicontinuous activity in the ripple band (80-250 Hz), which we defined continuous High Frequency Activity (HFA). We aim to analyze in all brain regions the occurrence and significance of this particular pattern. METHODS Twenty patients implanted in mesial temporal and neocortical areas were studied. One minute of slow-wave sleep was reviewed. The background was classified as continuous/semicontinuous, irregular, or sporadic based on the duration of the fast oscillations. Each channel was classified as inside/outside the seizure onset zone (SOZ) or a lesion. RESULTS The continuous/semicontinuous HFA occurred in 54 of the 790 channels analyzed, with a clearly higher prevalence in hippocampus and occipital lobe. No correlation was found with the SOZ or lesions. In the occipital lobe the continuous/semicontinuous HFA was present independently of whether eyes were open or closed. CONCLUSIONS We describe what appears to be a new physiological High Frequency Activity, independent of epileptogenicity, present almost exclusively in the hippocampus and occipital cortex but independent of the alpha rhythm. SIGNIFICANCE The continuous HFA may be an intrinsic characteristic of specific brain regions, reflecting a particular type of physiological neuronal activity.

Aminorex poisoning in cocaine abusers

Levamisole is found in more than 80% of illicit cocaine seized within United States borders. Percentages are somewhat lower in Europe. In 2009, controlled in vivo studies demonstrated that horses metabolize levamisole to aminorex. Earlier this year our laboratory demonstrated that the same conversion occurs in man. Levamisole itself causes aplastic anemia and numerous reports have begun to appear in the literature, but the conversion of levamisole to aminorex is of much more concern. Aminorex ingestion was responsible for a five-year epidemic (1967-1972) of idiopathic pulmonary hypertension (IPH) confined to Switzerland, Austria, and Germany, the only countries where aminorex had been marketed as an anorectic. The incidence of IPH reverted to normal levels as soon as aminorex was withdrawn. In most cases onset of symptoms in IPH began after six to nine months of aminorex use, with average dosage ranges of 10 to 40 mg per day. The outcome was almost uniformly fatal. The conversion rate of levamisole to aminorex has not been established, but given the high daily intake of cocaine by many abusers, it seems likely that many of them will have ingested enough contaminated cocaine to ultimately cause IPH. Until the disease is well established, the symptoms of IHP are vague, and existing drug registries specifically exclude drug abusers, making it difficult to track these cases. This review is intended to draw attention to what may be a slowly emerging new epidemic.

A mixed MDPV and benzodiazepine intoxication in a chronic drug abuser: Determination of MDPV metabolites by LC-HRMS and discussion of the case §

We report on a case of repeated MDPV consumptions that resulted in severe psychosis and agitation prompting the concomitant abuse of benzodiazepines. A 27-year-old man was found irresponsive in his apartment and was brought to the emergency department (ED) of a local hospital. When in ED, he rapidly recovered and self-reported to have recently injected some doses of MDPV that he had bought in the Internet. He left the hospital without medical cares. 15 days after, he was again admitted to the same ED due to severe agitation, delirium and hallucinations, and reported the use of MDPV and pharmaceutical drugs during the preceding week. He was sedated with diazepam and chlorpromazine. Urine samples collected in both occasions were sent for testing using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and liquid chromatography-high resolution multiple mass spectrometry (LC-HRMS/MS) on an Orbitrap. The LC-HRMS analysis revealed the presence of MDPV and its phase I and phase II metabolites (demethylenyl-MDPV, demethylenyl-methyl-MDPV, demethylenyl-methyl-oxo-MDPV, demethylenyl-hydroxy-alkyl-MDPV, demethylenyl-methyl-hydroxy alkyl-MDPV, demethylenyl-oxo-MDPV and their corresponding glucuronides), alprazolam and alprazolam metabolite at the first ED admission; at the time of the second ED access, the same MDPV metabolites, alprazolam, temazepam, and chlordiazepoxide were detected together with diazepam and metabolites. LC-HRMS/MS was use to determine the following concentrations, respectively on his first and second admission: MDPV 55ng/mL, alprazolam 114ng/mL, α-hydroxyalprazolam 104ng/mL; MDPV 35ng/mL, alprazolam 10.4ng/mL, α -hydroxyalprazolam 13ng/mL; chlordiazepoxide 13ng/mL, temazepam 170ng/mL, diazepam 1.3ng/mL, nordiazepam 61.5, oxazepam 115ng/mL. The toxicological findings corroborated the referred concomitant use of multiple pharmaceutical drugs and benzodiazepines. Confirmation of previous hypothesis on human metabolism of MDPV could be inferred by the analysis of urine.

Comparison of Immunoassay Screening Tests and LC–MS-MS for Urine Detection of Benzodiazepines and Their Metabolites: Results of a National Proficiency Test

For most diverse purposes, different immunoassay (IA) screening methods are usually used to detect benzodiazepines and their metabolites in urine. In this study, we compared the main IAs used in forensic toxicology (Cloned Enzyme Donor Immunoassay, CEDIA®; Enzyme-Multiplied Immunoassay Technique, EMIT®; Fluorescent Polarization ImmunoAssay, FPIA®; Kinetic Interaction of Microparticles in Solution, KIMS® and Immunochromatographic Techniques, IMC) with liquid chromatography-tandem mass spectrometry (LC-MS-MS). Twelve urine specimens were analyzed by 178 laboratories in Italy that participated in a National Proficiency Test, providing both qualitative and semi-quantitative results. Each IA was evaluated by the parameters: true positive, true negative, false positive (FP), false negative (FN), sensitivity (SENS), specificity (SPEC), positive predictive value, negative predictive value (NPV) and accuracy. SPEC was affected by a high FP rate for all IAs. The lowest SENS and NPV were provided by FPIA due to a high number of FN cases. Comparing IA semi-quantitative data with LC-MS-MS results, an overestimation of benzodiazepine amount is noted. This paper draws attention to the problem of the careless use of IA tests for forensic purposes as they may provide FP and/or FN results that can lead to errors of great severity.

Determination of fatty acid ethyl esters in hair by GC–MS and application in a population of cocaine users

A gas chromatography-mass spectrometry method for the determination of ethyl myristate, ethyl palmitate, ethyl oleate, and ethyl stearate in hair samples was developed, validated and applied to real samples. Ethyl myristate, ethyl palmitate, ethyl oleate, and ethyl stearate are fatty acid ethyl esters (FAEE) which are known to be direct biotransformation products of ethanol. Their presence in the body fluids and tissue is therefore indicative of alcohol intake and, in particular, FAEE concentration in hair higher than 0.5 ng/mg is indicative of excessive chronic alcohol consumption. The method was applied to 80 hair samples formerly found positive for cocaine and FAEE analytical results were compared with the presence of cocaethylene, a cocaine metabolite formed only when alcohol and cocaine are used together. According to our data the two biomarkers (FAEE and cocaethylene in hair) are tools of great value in the assessment of the diagnosis of use of cocaine and ethanol. In fact, discrepancies were noted and might be related to various factors including differences in consumption habits and thus permitting to distinguish the use of both substances non-concurrently or concurrently. Also, the determination of both markers may, in some cases, discriminate the use of moderate or heavy alcohol amounts when associated with cocaine. Finally, in a population of non-cocaine-users our results support FAEE as valuable means in the assessment of excessive alcohol chronic use.

What constitutes a normal ante-mortem urine GHB concentration?

Gamma-hydroxybutyric acid (GHB) is endogenously produced within the central nervous system, however it is also used as a medication for the treatment of a variety of clinical conditions, sold under the name Zyrem in the United States and Alcover in Europe. It is a very dangerous drug with a very limited safety margin, and is classified as a controlled substance in many countries. The interpretation of post-mortem studies of GHB concentrations is problematic; GHB can be detected in urine and blood from non-GHB users, both before and after death, and concentrations in both matrices may rise with prolonged storage. Because it is produced as a post-mortem artifact, forensically defensible cut-offs for post-mortem blood concentrations have yet to be established. Given the enormous degree of inter and intra-individual variation in GHB production that has been documented, it is unlikely they ever will. The important issue for forensic scientists is whether the detection of GHB in urine, in concentrations above some yet to be determined value, can be used as evidence for drug facilitated assault. In an attempt to see if a cut-off level could be determined we analyzed urine from 39 alcoholics who were being treated with known oral doses of Alcover (group 1), and compared the results with concentrations found in the urine of 30 volunteers who had no exogenous GHB intake (group 2), and 30 urine specimens taken from the alcoholics before they initiated GHB therapy (Alcover treatment group 3). More than one third (36.6%) of subjects being treated with GHB were found to have urinary GHB concentration that fell between 2.75 and 10 microg/mL. The data suggests that caution must be used when applying the currently used cut-off of 10 microg/mL.

Cross-reactivities and structure-reactivity relationships of six benzodiazepines to EMIT(®) immunoassay.

Benzodiazepines are among the most frequently prescribed drugs due to their sedative, hypnotic, anxiolytic, muscle relaxant and antiepileptic properties. Considering the high consumption of benzodiazepines worldwide, there is increased potential for addiction and abuse in cases of crime, driving under the influence of drugs, suicide and drug-facilitated sexual assault (DFSA). For these reasons, this class of drugs and their metabolites are frequently present in both clinical and forensic cases. In a forensic toxicology laboratory, typical screening analysis for benzodiazepine involves various immunoassay screening methods. The present study investigates the cross-reactivity profiles of six benzodiazepines not included in the manufacturers instructions (3-hydroxy-flunitrazepam, 7-amino-nitrazepam, brotizolam, delorazepam, pinazepam, α-hydroxy-midazolam) to EMIT(®) II Plus Benzodiazepine Assay. Pinazepam, delorazepam and brotizolam are the most reactive molecules, while the other ones present a very low cross-reactivity. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to confirm the concentrations of the spiked urines for immunoassay test and to make a comparison between the quantitative results of the different methods. Structure-reactivity relationships to EMIT(®) II Plus Benzodiazepine Assay were also evaluated. This paper draws attention to the problem of careless use of immunoassay tests for forensic purposes as they may provide false positive and/or negative results.