Human case of West Nile neuroinvasive disease in Portugal, 2015.

A case of West Nile virus (WNV) infection was reported in the Algarve region, Portugal, in the first week of September 2015. WNV is known to circulate in Portugal, with occasional reports in horses and birds (2004 to 2011) and very sporadically human cases (in 2004 and in 2010). Here we present the clinical and laboratory aspects related to the first human case of West Nile neuroinvasive disease reported in Portugal.

Sociodemographic, Clinical, and Behavioral Factors Associated with Sexual Transmitted Infection among HIV-1 Positive Migrants in Portugal: Are There Differences between Sexes?

INTRODUCTION: Sexually transmitted infections (STIs) continue to occur at high levels. According to the WHO, each year there are an estimated 374 million new infections with syphilis, gonorrhea, chlamydia, and trichomoniasis. STIs are associated with an increased risk of acquiring HIV infection. Migrants are reportedly highly affected by STIs. OBJECTIVES: This study aims to characterize factors associated with STIs in a population of HIV-positive migrants living in Portugal. METHODOLOGY: This is a cross-sectional observational study of 265 newly diagnosed HIV-1 positive migrants, who were defined as individuals born outside Portugal. This group of people were part of the BESTHOPE study that was developed in 17 Portuguese hospitals between September 2014 and December 2019, and included information collected through sociodemographic and behavioral questionnaires filled in by the migrant patients, clinical questionnaires filled in by the clinicians and HIV-1 genomic sequences generated through resistance testing (Sanger sequencing). A multivariable statistical analysis was used to analyze the association between sociodemographic characteristics, sexual behaviors, HIV testing and sexual infections. RESULTS: Most HIV-1 positive individuals included in the study were men (66.8%) and aged between 25 and 44 years old (59.9%). Men had a higher proportion of STIs when compared to women (40.4% vs. 14.0%) and the majority of men reported homosexual contacts (52.0%). Most men reported having had two or more occasional sexual partners in the previous year (88.8%) and 50.9% reported always using condoms with occasional partners, while 13.2% never used it. For regular partners, only 29.5% of the women reported using condoms, compared to 47.3% of men. Other risk behaviors for acquiring HIV, such as tattooing and performing invasive medical procedures, were more prevalent in men (38.0% and 46.2%, respectively), when compared to women (30.4% and 45.1% respectively) and 4.7% of men reported having already shared injectable materials, with no data for comparison in the case for women. Additionally, 23.9% of women reported having had a blood transfusion while only 10.3% of men reported having had this medical procedure. Meanwhile, 30.9% of the individuals reported having been diagnosed with some type of STI in the last 12 months. In addition, 43.3% of individuals that answered a question about hepatitis reported to be infected with hepatitis B, while 13.0% reported having hepatitis C infection. According to the multivariable analysis, the only transmission route was significantly associated with reports of previous STI infection: men who have sex with men (MSM) were 70% more likely to have been diagnosed with an STI in the past 12 months compared to the heterosexual route. CONCLUSION: HIV-1 infected men were more likely to report previous STIs than women. On the other hand, most migrant women had a regular sexual partner and never or only sometimes used condoms. This somewhat discrepant findings suggest that gender inequalities may make women unable to negotiate safe sexual practices, resulting in increased susceptibility to infection. However, since migrant women report less STIs, we cannot exclude that these STIs may remain undiagnosed. The implementation of safer sex awareness campaigns for condom use and screening for STIs in women is crucial. On the other hand, health education campaigns for STI knowledge need to be implemented for both MSM and women and their partners.

Determinants of HIV late presentation among men who have sex with men in Portugal (2014-2019): who's being left behind?

Introduction: HIV late presentation (LP) remains excessive in Europe. We aimed to analyze the factors associated with late presentation in the MSM population newly diagnosed with HIV in Portugal between 2014 and 2019. Methods: We included 391 newly HIV-1 diagnosed Men who have Sex with Men (MSM), from the BESTHOPE project, in 17 countrywide Portuguese hospitals. The data included clinical and socio-behavioral questionnaires and the viral genomic sequence obtained in the drug resistance test before starting antiretrovirals (ARVs). HIV-1 subtypes and epidemiological surveillance mutations were determined using different bioinformatics tools. Logistic regression was used to estimate the association between predictor variables and late presentation (LP). Results: The median age was 31 years, 51% had a current income between 501-1,000 euros, 28% were migrants. 21% had never been tested for HIV before diagnosis, with 42.3% of MSM presenting LP. 60% were infected with subtype B strains. In the multivariate regression, increased age at diagnosis, higher income, lower frequency of screening, STI ever diagnosed and higher viral load were associated with LP. Conclusion: Our study suggests that specific subgroups of the MSM population, such older MSM, with higher income and lower HIV testing frequency, are not being targeted by community and clinical screening services. Overall, targeted public health measures should be strengthened toward these subgroups, through strengthened primary care testing, expanded access to PrEP, information and promotion of HIV self-testing and more inclusive and accessible health services.

LC-MS-MS-MS3 for the determination and quantification of ∆9-tetrahydrocannabinol and metabolites in blood samples.

Due to the high prevalence of cannabinoids in forensic toxicology analysis, it is crucial to have an efficient method that allows the use of a small sample amount and that requires a minimal sample preparation for the determination and quantification of low concentrations. A simple, highly selective and high throughput liquid chromatography-tandem mass spectrometry methodology (LC-MS-MS-MS3) was developed for the determination and quantification of ∆9-tetrahydrocannabinol (THC), 11-hydroxy-∆9- tetrahydrocannabinol (THC-OH) and 11-nor-9-carboxy-∆9-tetrahydrocannabinol (THC-COOH) in blood samples. Chromatographic analysis of THC, THC-OH and THC-COOH and their deuterated internal standards was preceded by protein precipitation (PPT) of 0.1 mL of blood samples with acetonitrile. Chromatographic separation was achieved by use of an Acquity UPLC® HHS T3 (100 mm × 2.1 mm i.d., 1.8 µm) reversed-phase column, using a gradient elution of 2 mM aqueous ammonium formate, 0.1% formic acid and methanol at a flow rate of 0.4 mL/min, with a run time of 10 min. For the MS-MS-MS3 analysis, a SCIEX QTRAP® 6500+ triple quadrupole linear ion trap mass spectrometer was used via electrospray ionization (ESI), operated in multiple reaction monitoring (MRM) and linear ion trap mode (MS3). The method was validated in accordance with internationally accepted criteria and guidelines, and proved to be selective and linear between 0.5 and 100 ng/mL (r2 > 0.995). The lower limits of quantification (LLOQ) corresponded to the lowest concentrations used for the calibration curves. The coefficients of variation obtained for accuracy and precision were <15%. The mean recoveries were between 88.0% and 117.2% for the studied concentration levels (1 ng/mL, 5 ng/mL and 50 ng/mL). No significant interfering compounds, matrix effects or carryover were observed. The validated method provides a sensitive, efficient and robust procedure for the quantification of cannabinoids in blood, using LC-MS-MS-MS3 and a sample volume of 0.1 mL. This work is also a proof of concept for using LC-MS3 technique to determine drugs in biological samples.

HIV-1-Transmitted Drug Resistance and Transmission Clusters in Newly Diagnosed Patients in Portugal Between 2014 and 2019.

Objective: To describe and analyze transmitted drug resistance (TDR) between 2014 and 2019 in newly infected patients with HIV-1 in Portugal and to characterize its transmission networks. Methods: Clinical, socioepidemiological, and risk behavior data were collected from 820 newly diagnosed patients in Portugal between September 2014 and December 2019. The sequences obtained from drug resistance testing were used for subtyping, TDR determination, and transmission cluster (TC) analyses. Results: In Portugal, the overall prevalence of TDR between 2014 and 2019 was 11.0%. TDR presented a decreasing trend from 16.7% in 2014 to 9.2% in 2016 (p for-trend = 0.114). Multivariate analysis indicated that TDR was significantly associated with transmission route (MSM presented a lower probability of presenting TDR when compared to heterosexual contact) and with subtype (subtype C presented significantly more TDR when compared to subtype B). TC analysis corroborated that the heterosexual risk group presented a higher proportion of TDR in TCs when compared to MSMs. Among subtype A1, TDR reached 16.6% in heterosexuals, followed by 14.2% in patients infected with subtype B and 9.4% in patients infected with subtype G. Conclusion: Our molecular epidemiology approach indicates that the HIV-1 epidemic in Portugal is changing among risk group populations, with heterosexuals showing increasing levels of HIV-1 transmission and TDR. Prevention measures for this subpopulation should be reinforced.

Identification and Quantification of Antipsychotics in Blood Samples by LC-MS-MS: Case Reports and Data from Three Years of Routine Analysis.

Antipsychotic drugs (AP) are widely prescribed for the treatment of schizophrenia and psychosis. The pharmacological treatment of schizophrenia is often performed with the simultaneous use of two or more antipsychotic agents to achieve the desired control of psychotic symptoms Available AP include both conventional (typical) and new (atypical) antipsychotic medications. Atypical AP, such as quetiapine, now account for the vast majority of AP prescriptions. In forensic toxicology, AP are of considerable interest because of their potential abuse and their involvement in intoxications and suicides. The authors retrospectively examined AP positive cases detected in samples collected during autopsies performed in the Forensic Clinical and Pathology Service of National Institute of Legal Medicine and Forensic Sciences Centre Branch or in other autopsies carried out in the central region of Portugal, between January 2016 and December 2018. A quantitative liquid chromatography-tandem mass spectrometry assay was developed for the simultaneous determination of 16 AP (amisulpride, aripiprazole, chlorpromazine, clozapine, cyamemazine, fluphenazine, haloperidol, levomepromazine, melperone, olanzapine, paliperidone, promethazine, quetiapine, risperidone, sulpiride and ziprasidone) in blood samples of postmortem cases. The Laboratory of Forensic Chemistry and Toxicology received 3,588 requests for toxicological analysis: 1,413 cases were positive for drugs from which 351 (24.8%) cases were positive for AP, 60.1% from male individuals and 39.9% from female. Quetiapine was the most prevalent AP (36.5%) followed by olanzapine (20.8%). During this period, there were 25 postmortem cases with AP blood concentrations above therapeutic range, in which 36% of those are in agreement with the information received (psychological history or acute intoxication suspicion) and the manner of death was suicide. Our results point that antipsychotics are an increasingly prevalent class of drugs. AP must be measured not only in toxic concentrations but also in therapeutic levels in postmortem cases; therefore, it is important to come up with a sensitive method to cover the low therapeutic range in which AP are usually present.

The presence of eschars, but not greater severity, in Portuguese patients infected with Israeli spotted fever.

In Portugal, Mediterranean spotted fever (MSF) is caused by R. conorii Malish and Israeli spotted fever (ISF) strains. It has been suggested that the ISF strain isolated from patients with MSF causes different clinical manifestations compared to those caused by Malish strain, namely the absence of eschar and greater severity. The aim of this study was to analyze the presence or absence of eschar and of fatality in Portuguese patients infected with either Malish or ISF strain. Of 94 patients with a clinical diagnosis of MSF between 1994 to 2004, 47 were infected with Malish strain and 47 with ISF strain. Eschars were reported in 20 patients (49%) infected with Malish strain, and in 17 (39%) with ISF strain. The presence of eschar is not statistically associated to a greater extent with either R. conorii strain (P=0.346). A total of 22 patients died, 9 infected with Malish strain and 13 infected with ISF strain, and no statistically significant difference was found (P=0.330). This study showed that the concepts of absence of the eschar and greater severity in Israeli spotted fever infection should be revised.

Analysis of Delta9-tetrahydrocannabinol in oral fluid samples using solid-phase extraction and high-performance liquid chromatography-electrospray ionization mass spectrometry.

An analytical method using solid-phase extraction (SPE) and high-performance liquid chromatography-mass spectrometry (LC-MS) has been developed and validated for the confirmation of Delta(9)-tetrahydrocannabinol (THC) in oral fluid samples. Oral fluid was extracted using Bond Elut LRC-Certify solid-phase extraction columns (10 cm(3), 300 mg) and elution performed with n-hexane/ethyl acetate. Quantitation made use of the selected ion-recording mode (SIR) using the most abundant characteristic ion [THC+H(+)], m/z 315.31 and the fragment ion, m/z 193.13 for confirmation, and m/z 318.00 for the protonated internal standard, [d(3)-THC+H(+)]. The method proved to be precise for THC, in terms of both intra-day and inter-day analyses, with coefficients of variation less than 10%, and the calculated extraction efficiencies for THC ranged from 76 to 83%. Calibration standards spiked with THC between 2 and 100 ng/mL showed a linear relationship (r(2)=0.999). The method presented was applied to the oral fluid samples taken from the volunteers during the largest music event in Portugal, named Rock in Rio-Lisboa. Oral fluid was collected from 40 persons by expectoration and with Salivette. In 55% of the samples obtained by expectorating, THC was detected with concentration ranges from 1033 to 6552 ng/mL and in 45% of cases THC was detected at concentrations between 51 and 937 ng/mL. However, using Salivette collection, 26 of the 40 cases had an undetectable THC.

Two fatal intoxication cases with imidacloprid: LC/MS analysis.

Imidacloprid [1-(6-chloro-3pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine] is a new and potent nitromethylene insecticide with high insecticidal activity at very low application rates. It is the first highly effective insecticide that, like nicotine, acts on the nervous system, causing blockage of postsynaptic nicotinergic acetylcholine receptors. Two fatal cases with this insecticide in two male individuals, of 33 and 66 years old, are presented. An LC/MS with electrospray method for measuring imidacloprid and its metabolites in post-mortem samples is described. In the chromatographic separation, a reverse-phase column XTerra MS C18 (2.1mm i.d.x 150 mm, 5 microm) was used and the mobile phase composed with acetonitrile and 0.1% formic acid (15:85), at a 0.25 mL/min flow rate. Samples were prepared with a liquid-liquid extraction procedure with dichloromethane. Calibration curves for imidacloprid in blood and urine samples were linear from 0.2 to 15 microg/mL. The mean recovery was 86% with a coefficient of variation of +/-5.9%. The detection limit was 0.002 microg/mL. Quantitative results were obtained for all post-mortem matrices available of the two fatal cases: blood, urine, stomach contents, lung, liver and kidney. The imidacloprid blood concentrations found in two-cases were 12.5 and 2.05 microg/mL. The authors validated a method to detect and quantify imidacloprid in post-mortem samples, and to our knowledge for the first time a post-mortem tissue distribution was performed on various samples for this insecticide.

Pesticide intoxications in the Centre of Portugal: three years analysis.

Pesticides are used in most countries around the world to protect agricultural and horticultural crops against damage. Poisoning by these toxicant agents occurs as a result of misuse or accidental exposure, and also by oral ingestion (voluntary or not). In Portugal, pesticide intoxications are still a cause of death, found in a considerable number of cases. The authors retrospectively examined the cases of pesticide poisoning in the Centre of Portugal, from autopsies performed in the Forensic Pathology Service of Coimbra's Delegation of the National Institute of Legal Medicine (NILM) and from other autopsies carried out in the Centre of Portugal, as well as some samples taken in hospitals in cases of suspected intoxication. In this study, the positive cases have been especially studied, in order to identify the pesticide used, as well as the etiology. The frequency of intoxications and its distribution by sex and age were also analyzed. Between January 2000 and December 2002, the Forensic Toxicology Laboratory received 639 pesticide analysis requests. In 2000, in a total of 149 analysis requests, 30 cases were positive, 63.3% from male individuals and 36.7% from female. In 2001, the analysis requests increased to 240 as well as the positive cases (43), 74.4% from male individuals and 25.6% from female and in 2002, the total cases analyzed also increased to 250, with 38 positive (73.6% from male individuals and 26.4% from female). Among the pesticides, organophosphorus insecticides still constitute the most important class detected in forensic intoxications, representing 63% of the total positive cases, followed by herbicides, with 33% of the positive results. Quinalphos is the most important organophosphorus insecticide, present in 32 of the 111 positive cases, followed by the herbicide paraquat, detected in 31 cases. The study emphasizes the increasing number of pesticide analyses, particularly relevant for the organophosphorus compounds and herbicides. Intoxication suspicion, accidental or voluntary, seems to be the most common cause of the incidents, for which analyses are requested, but it is also evident that the putative cause is unknown in a large number of cases. Therefore, more stringent legislation and enforcement regarding the sale and distribution of these toxic substances are needed.

Forensic intoxication with clobazam: HPLC/DAD/MSD analysis.

Clobazam (Castillium, Urbanil), a benzodiazepine often used as an anxiolytic and in the treatment of epilepsy, is considered a relatively safe drug. The authors present a fatal case with a 49-year-old female, found dead at home. She had been undergoing psychiatric treatment and was a chronic alcoholic. The autopsy findings were unremarkable, except for multivisceral congestion, steatosis and a small piece of a plastic blister pack in the stomach. Bronchopneumonia, bronchitis and bronchiolitis were also diagnosed. Anhigh-performance liquid chromatography (HPLC)/diode array detector (DAD)/mass spectrometry detection (MSD) with electrospray method was developed in order to detect, confirm and quantify clobazam in the post-mortem samples. In the chromatographic separation, a reversed-phase column C18 (2.1 x 150 mm, 3.5 microm) was used with a mobile phase of methanol and water, at a 0.25 ml/min flow rate. Carbonate buffer (pH 10.5) and 20 microl of prazepam (100 microg/ml) as internal standard were added to the samples. A simple and reliable liquid-liquid extraction method for the determination of clobazam in post-mortem samples was described. Calibration curves for clobazam were performed in blood, achieving linearity between 0.01 and 10 microg/ml and a detection limit of 1.0 ng/ml. The clobazam concentration found in post-mortem blood was 3.9 microg/ml, higher than the reported therapeutic concentration (0.1-0.4 microg/ml). The simultaneous acquisition by photodiode array detection and mass spectrometry detection results allowed benzodiazepines to be identified with sufficient certainty. An examination of all the available information suggested that death resulted from respiratory depression due to clobazam toxicity.

Cannabis and driving: the use of LC-MS to detect delta9-tetrahydrocannabinol (delta9-THC) in oral fluid samples.

Cannabis is the most widely used illicit drug in the world. There is strong evidence from performance studies that delta9-THC has significant effects on the cognitive and psychomotor tasks associated with driving. New, easy and sensitive methods to detect impaired drivers are needed. Therefore, it is necessary the use of alternative biological samples which may be accurate, precise and with trustful interpretation results. This article presents an LC-MS methodology for detecting delta9-tetrahydrocannabinol (delta9-THC) in oral fluid samples. The mean recovery was 79%, coefficients of variations were between 2.9% and 6.9% and the limits of detection (LOD) and quantitation (LOQ) were 1.0 ng/ml and 2.0 ng/ml, respectively. The method is sensitive, accurate and reproducible and may be utilized in ongoing controlled cannabinoid administration studies and in roadside studies and thus, important for the fields of forensic toxicology.

Aldicarb poisoning: one case report.

The carbamate aldicarb (Temik) is a highly toxic (LD50 = 0.9 mg/kg oral in rats) insecticide often used in agriculture. The authors present a fatal intoxication case with aldicarb, in a 24-year-old male, under police custody in the island of S. Tome and Prince (STP), in Africa. He was found 3 h after his arrest, lying on the floor of his cell, unconscious, frothing at the mouth, and entered into the hospital already dead. Aldicarb analysis was carried out by high-performance liquid chromatography, with a post-column derivatization system (with o-phthaldialdehyde and 2-mercaptoethanol, under alkaline conditions at 80 degrees C) with fluorescence detection at lambda(ex)= 339 nm and lambda(em) = 445 nm. The toxicological analysis revealed toxic concentrations of aldicarb in the post-mortem samples: blood (6.2 microg/ml), stomach (48.9 microg/g), liver (0.80 microg/g), kidney (8.10 microg/g), heart (6.70 microg/g) and urine (17.50 microg/ml). It was also detected to be the same substance in a grey granulate powder supplied by criminal authorities of that country, as a probable substance ingested by the victim. The toxicological analysis results, the autopsy findings and the information given, allowed us to conclude that death occurred due to an aldicarb acute intoxication. Although the case suggested a suicide, particularly based on the known information and on the high aldicarb concentration found in the stomach, it was not possible to indicate precisely its etiology (suicide or homicide).

Validated UPLC-MS/MS assay for the determination of synthetic phosphodiesterase type-5 inhibitors in postmortem blood samples.

The use of synthetic phosphodiesterase type 5 (PDE-5) inhibitors for the treatment of erectile dysfunction: sildenafil citrate (Viagra(®)), tadalafil (Cialis(®)) and vardenafil hydrochloride (Levitra(®)) has increased dramatically over the past 2 years. These substances are prescription drugs and must be used under medical supervision. However, they can easily be obtained over the internet from illegal sites, being a potential for a threat to public health. The development of an electrospray ionisation (ESI) ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) procedure for the simultaneous identification and quantification of three PDE5 inhibitors in blood samples was desired. Samples were prepared using Oasis(®) HLB solid-phase cartridges (3 cc, 60 mg) and chromatographic separation was achieved on an Acquity UPLC(®) HSS T3 (100 × 2.1 mm i.d., 1.8 µm particles) column with a gradient mobile phase of 0.1% formic acid and acetonitrile at a 0.5 mL/min flow rate. Quantification was achieved by multiple reaction monitoring (MRM) of two transitions per compound: m/z 475.1 > 58 e m/z 475.1 > 311.1 for sildenafil; m/z 389.9 > 267.9 e m/z 389.9 > 134.8 for tadalafil and m/z 489 > 71.9 e m/z 489 > 150.9 for vardenafil. Zolpidem-d6 (m/z 314.5 > 235.3) was used as the internal standard. Calibration curves were linear over the concentration range of 5-1000 ng/mL, with a coefficient of determination better than 0.997. The lower limits of detection and quantification for these substances were ≤ 3 ng/mL and ≤ 8 ng/mL, respectively. The method showed a satisfactory sensitivity, precision, accuracy, recovery and selectivity. A rapid, selective and sensitive UPLC-MS/MS method using solid-phase extraction was developed for the simultaneous determination and quantification of sildenafil, vardenafil and tadalafil in blood samples.

UPLC-MS/MS determination in blood of a mixed-drug fatal intoxication: a case report.

Trends in forensic toxicology show the introduction of rapid analytical methods for the simultaneous quantitative analysis of drugs. The authors present a fatal case involving a 32-year-old male, found dead in bed by his mother, with several blue, white and orange pills next to the body. Empty tablets were found in the trash bin and a suicide note was on the desk. He was diagnosed with bipolar disorder and had been under psychiatric treatment, having repeatedly demonstrated intent to commit suicide. A rapid method was developed to determine 55 different medicines and 32 benzodiazepines in blood by ultra-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS) with electrospray ionization source in positive and negative ion mode. Chromatographic analysis was preceded by an optimized solid-phase extraction procedure using Oasis(®) HLB (3 cc, 60 mg) extraction columns. The extracted analytes were separated by UPLC (Waters) with a reversed-phase Acquity UPLC(®) HSS T3 (2.1×100 mm id, 1.8 µm) column with acetonitrile and 0.1% formic acid in water as mobile phase, at 0.5 mL/min flow rate and a chromatographic run-time of 8 min. Analytes detection was achieved with a triple-quadrupole mass spectrometer in positive and negative electrospray ionization mode with multiple reaction monitoring (MRM). Two MRM transitions were monitored for each target-compound and one for each deuterated internal standards. Toxicological results showed high blood concentrations of antipsychotics (haloperidol, olanzapine and quetiapine), antidepressants (fluoxetine and paroxetine) and anxiolytics (bromazepam and lorazepam). Risperidone and other benzodiazepines were also present in therapeutic concentrations. Neither alcohol nor illicit drugs were present in the analyzed samples. The UPLC-MS-MS method showed to be appropriate for screening, identification and quantitation of antipsychotics, antidepressants, anxiolytics and antiepileptic drugs in blood after intake of therapeutic as well as toxic doses. The autopsy and toxicological results led the pathologist to rule that death was due to a mixed-drug intoxication. The manner of death was determined to be suicide.

An UPLC-MS/MS method for the determination of valproic acid in blood of a fatal intoxication case.

Valproic acid (VPA) has been used as an anticonvulsant for the treatment of epilepsy. The authors present a fatal case involving a 45-year-old female, found dead lying in bed with empty tablets of Diplexil(®) next to her. She was a chronic alcoholic and epileptic who had been under psychiatric treatment, having repeatedly demonstrated intent to commit suicide. A rapid method was developed and validated to determine VPA in blood by ultra-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS) with electrospray ionization source in negative ion mode. The method involved sample treatment with phosphoric acid followed by solid-phase extraction. Chromatographic separation was achieved using an Acquity UPLC(®) BEH (2.1 × 50 mm id, 1.7 µm) column and a mobile phase containing ammonium acetate and acetonitrile, at a 0.5 mL/min flow rate. Detection and quantification of VPA was achieved using multiple reaction monitoring (MRM). The MS/MS transitions used for monitoring were m/z 143.1-143.1 for valproic acid and m/z 296.1-205.0 for hydrochlorothiazide used as an internal standard (IS). The limit of quantification (LOQ) was 0.5 µg/mL and the method was linear in the concentration range of 0.5-100 µg/mL. The coefficients of variation obtained for accuracy and precision were less than 10% and the mean recovery was 95% for the three concentrations levels studied (5 µg/mL, 10 µg/mL and 50 µg/mL). Toxicological results showed high concentration of VPA (556 µg/mL) and therapeutic concentrations of tiapride, mirtazapine, oxazepam and nordiazepam. Blood sample analysis also revealed the presence of ethanol at a concentration of 1.34 g/L. A specific, selective and sensitive method for the determination of VPA in blood was developed and can be used in routine forensic investigation. Toxicological results led the pathologist to rule that death was due to an intoxication caused by the simultaneous ingestion of high VPA concentrations and alcohol, with a suicidal legal-medical etiology.

[Toscana virus in the Portuguese population: serosurvey and clinical cases]. / Vírus Toscana na população Portuguesa: vigilância sero-epidemiológica e casos clínicos.

Toscana virus (Phlebovirus genus, Bunyaviridae family) is a neurotropic virus which circulates in the Mediterranean Basin. Although Portugal has been the second country where its presence was reported, the existence of this virus in our country has been referred only sporadically, and there is a lack of knowledge regarding the prevalence of antibodies in the population. Thus, the objective of this study was to analyse the prevalence of antibodies anti-Toscana virus in the human population in our country. Sero-epidemiological investigations were performed with indirect immunofluorescence assay (IFA) and enzyme linked immunosorbent assay (ELISA) tests. The study population consisted of a control population (blood donors, n=150), a population considered at risk (n=236) and a population of individuals with symptoms and laboratory diagnostic request for vector-borne viruses. The latter population was divided into two groups: those individuals with neurological symptoms (n=165) and those without neurological symptoms (n=373). We tested sera from a total of 924 individuals. The seroprevalence of IgG antibodies in the control population was 2%. In the population considered at risk, the prevalence was 3.4%. In the population with central nervous system disease, we detected a seroprevalence of 4.2%. For the same type of antibodies and in subjects without central nervous system disease, the prevalence was 1.3%. Five cases of recent infection (3%) were detected in the population with neurological signs. Those infections have been acquired in the districts of Faro, Coimbra, Aveiro and Lisbon. The associated clinical diagnoses were meningitis, meningoencephalitis and rash. The observed seroprevalences were, in general, lower than reported in other endemic countries. Only 5 of the 29 sera which gave positive results by IFA and ELISA were confirmed by plaque reduction neutralization tests with the Italian strain ISS.Phl.3. This can indicate the presence of more than one Toscana virus serotype circulating in Portugal and emphasizes the need for more research about this etiological agent in our country.

Host- and microbe-related risk factors for and pathophysiology of fatal Rickettsia conorii infection in Portuguese patients.

BACKGROUND: The pathophysiologic mechanisms that determine the severity of Mediterranean spotted fever (MSF) and the host-related and microbe-related risk factors for a fatal outcome are incompletely understood. METHODS: This prospective study used univariate and multivariate analyses to determine the risk factors for a fatal outcome for 140 patients with Rickettsia conorii infection admitted to 13 Portuguese hospitals during 1994-2006 with documented identification of the rickettsial strain causing their infection. RESULTS: A total of 71 patients (51%) were infected with the Malish strain of Rickettsia conorii, and 69 (49%) were infected with the Israeli spotted fever (ISF) strain. Patients were admitted to the intensive care unit (40 [29%]), hospitalized as routine inpatients (95[67%]), or managed as outpatients (5[4%]). Death occurred in 29 adults (21%). A fatal outcome was significantly more likely for patients infected with the ISF strain, and alcoholism was a risk factor. The pathophysiology of a fatal outcome involved significantly greater incidence of petechial rash, gastrointestinal symptoms, obtundation and/or confusion, dehydration, tachypnea, hepatomegaly, leukocytosis, coagulopathy, azotemia, hyperbilirubinemia, and elevated levels of hepatic enzymes and creatine kinase. Some, but not all, of these findings were observed more often in ISF strain-infected patients. CONCLUSIONS: Although fatalities and similar clinical manifestations occurred among both groups of patients, the ISF strain was more virulent than the Malish strain. Multivariate analysis revealed that acute renal failure and hyperbilirubinemia were most strongly associated with a fatal outcome.

Fatal intoxication with tianeptine (Stablon).

Tianeptine (Stablon), although structurally similar to tricyclic antidepressants, acts by enhancing the reuptake of serotonin. A fatal case is presented involving a 26-year-old man, found lying in bed with a "mushroom of foam" around his mouth. Empty blister packs of Stablon and a suicide note were found next to the body. A liquid-liquid extraction procedure with n-hexane: ethyl acetate and n-hexane: 2-propanol, followed by LC-DAD-MS analysis, using positive mode electrospray ionization was performed. The detection limit was 0.001 microg/mL. The toxicological results revealed the following tianeptine concentrations in the post-mortem samples: blood 5.1 microg/mL; urine 2.0 microg/mL; liver 23 microg/g; stomach contents 22 mg. Femoral blood analyses also revealed an ethanol concentration of 0.53 g/L. The present method was also developed and validated for the other post-mortem specimens, since no previous published data had confirmed the post-mortem distribution of tianeptine. The absence of other suitable direct causes of death (macroscopic or histological) and the positive results achieved with the toxicological analysis led the pathologist to rule that death was due to an intoxication caused by the suicidal ingestion of tianeptine in combination with alcohol.