Detection of hepatitis A, B, and C virus-specific antibodies using oral fluid for epidemiological studies

In this report, we examine the adaptability of commercially available serological kits to detect antibodies markers for viral hepatitis in oral fluid samples. We also assessed the prevalence of hepatitis A, B, and C virus-specific antibodies, and related risk factors for these infectious diseases through sensitivity of the tests in saliva samples to evaluate if oral fluid can be an alternative tool to substitute serum in diagnosis of acute viral hepatitis and in epidemiological studies. One hundred and ten paired serum and saliva specimens from suspect patients of having acute hepatitis were collected to detect antibodies to hepatitis A (total and IgM), hepatitis B (anti-HBs, total anti-HBc and IgM anti-HBc), and hepatitis C (anti-HCV) using commercially available enzyme-linked immunossorbent assay (EIA). In relation to serum samples, oral fluid assay sensitivity and specificity were as follows: 87 and 100% for total anti-HAV, 79 and 100% for anti-HAV IgM, 6 and 95% for anti-HBs, 13 and 100% for total anti-HBc, 100 and 100% for anti-HBc IgM, and 75 and 100% for anti-HCV. The consistency observed between antibodies tests in saliva and expected risk factors for hepatitis A and C suggests that the saliva method could replace serum in epidemiological studies for hepatitis A and C.

Analysis of cannabinoids in oral fluid by liquid chromatography-tandem mass spectrometry

A sensitive method was developed for quantifying a wide range of cannabinoids in oral fluid (OF) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). These cannabinoids include a dagger(9)-tetrahydrocannabinol (THC), 11-hydroxy-a dagger(9)-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-a dagger(9)-tetrahydrocannabinol (THCCOOH), cannabinol (CBN), cannabidiol (CBD), a dagger(9)-tetrahydrocannabinolic acid A (THC-A), 11-nor-9-carboxy-a dagger(9)-tetrahydrocannabinol glucuronide (THCCOOH-gluc), and a dagger(9)-tetrahydrocannabinol glucuronide (THC-gluc). Samples were collected using a Quantisal (TM) device. The advantages of performing a liquid-liquid extraction (LLE) of KCl-saturated OF using heptane/ethyl acetate versus a solid-phase extraction (SPE) using HLB copolymer columns were determined. Chromatographic separation was achieved in 11.5 min on a Kinetex (TM) column packed with 2.6-mu m core-shell particles. Both positive (THC, 11-OH-THC, CBN, and CBD) and negative (THCCOOH, THC-gluc, THCCOOH-gluc, and THC-A) electrospray ionization modes were employed with multiple reaction monitoring using a high-end AB Sciex API 5000 (TM) triple quadrupole LC-MS/MS system. Unlike SPE, LLE failed to extract THC-gluc and THCCOOH-gluc. However, the LLE method was more sensitive for the detection of THCCOOH than the SPE method, wherein the limit of detection (LOD) and limit of quantification (LOQ) decreased from 100 to 50 pg/ml and from 500 to 80 pg/ml, respectively. The two extraction methods were successfully applied to OF samples collected from volunteers before and after they smoked a homemade cannabis joint. High levels of THC were measured soon after smoking, in addition to significant amounts of THC-A. Other cannabinoids were found in low concentrations. Glucuronide conjugate levels were lower than the method's LOD for most samples. Incubation studies suggest that glucuronides could be enzymatically degraded by glucuronidase prior to OF collection

Comparison of cannabinoid concentrations in oral fluid and whole blood between occasional and regular cannabis smokers prior to and after smoking a cannabis joint.

A cross-over controlled administration study of smoked cannabis was carried out on occasional and heavy smokers. The participants smoked a joint (11 % Δ9-tetrahydrocannabinol (THC)) or a matching placebo on two different occasions. Whole blood (WB) and oral fluid (OF) samples were collected before and up to 3.5 h after smoking the joints. Pharmacokinetic analyses were obtained from these data. Questionnaires assessing the subjective effects were administered to the subjects during each session before and after the smoking time period. THC, 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THCCOOH) were analyzed in the blood by gas chromatography or liquid chromatography (LC)-tandem mass spectrometry (MS/MS). The determination of THC, THCCOOH, cannabinol (CBN), and Δ9-tetrahydrocannabinolic acid A (THC-A) was carried out on OF only using LC-MS/MS. In line with the widely accepted assumption that cannabis smoking results in a strong contamination of the oral cavity, we found that THC, and also THC-A, shows a sharp, high concentration peak just after smoking, with a rapid decrease in these levels within 3 h. No obvious differences were found between both groups concerning THC median maximum concentrations measured either in blood or in OF; these levels were equal to 1,338 and 1,041 μg/L in OF and to 82 and 94 μg/L in WB for occasional and heavy smokers, respectively. The initial WB THCCOOH concentration was much higher in regular smokers than in occasional users. Compared with the occasional smokers, the sensation of confusion felt by the regular smokers was much less while the feeling of intoxication remained almost unchanged.

Detection of Paraquat in Oral Fluid, Plasma, and Urine by Capillary Electrophoresis for Diagnosis of Acute Poisoning

FAPESP

Monitoring of threo-methylphenidate enantiomers in oral fluid by capillary electrophoresis with head-column field-amplified sample injection

Threo-methylphenidate is a chiral psychostimulant drug widely prescribed to treat attention-deficit hyperactivity disorder in children and adolescents. An enantioselective CE-based assay with head-column field-amplified sample stacking for analysis of threo-methylphenidate enantiomers in liquid/liquid extracts of oral fluid is described. Analytes are electrokinetically injected across a short water plug placed at the capillary inlet and become stacked at the interface between plug and buffer. Enantiomeric separation occurs within a few minutes in a pH 3.0 phosphate/triethanolamine buffer containing 20 mg/mL (2-hydroxypropyl)-β-CD as chiral selector. The assay with six point multilevel internal calibration provides a linear response for each enantiomer in the 10-200 ng/mL concentration range, is simple, inexpensive, and reproducible, and has an LOQ of 5 ng/mL. It was applied to oral fluid patient samples that were collected up to 12 h after intake of an immediate release tablet and two different extended release formulations with racemic methylphenidate. Drug profiles could thereby be assessed in a stereoselective way. Almost no levorotary threo-methylphenidate enantiomer was detected after intake of the two extended release formulations, whereas this enantiomer was detected during the first 2.5 h after intake of the immediate release preparation. The noninvasive collection of oral fluid is an attractive alternative to plasma for the monitoring of methylphenidate exposure in the pediatric community.

Detection of African Swine Fever Virus Antibodies in Serum and Oral Fluid Specimens Using a Recombinant Protein 30 (p30) Dual Matrix Indirect ELISA

In the absence of effective vaccine(s), control of African swine fever caused by African swine fever virus (ASFV) must be based on early, efficient, cost-effective detection and strict control and elimination strategies. For this purpose, we developed an indirect ELISA capable of detecting ASFV antibodies in either serum or oral fluid specimens. The recombinant protein used in the ELISA was selected by comparing the early serum antibody response of ASFV-infected pigs (NHV-p68 isolate) to three major recombinant polypeptides (p30, p54, p72) using a multiplex fluorescent microbead-based immunoassay (FMIA). Non-hazardous (non-infectious) antibody-positive serum for use as plate positive controls and for the calculation of sample-to-positive (S:P) ratios was produced by inoculating pigs with a replicon particle (RP) vaccine expressing the ASFV p30 gene. The optimized ELISA detected anti-p30 antibodies in serum and/or oral fluid samples from pigs inoculated with ASFV under experimental conditions beginning 8 to 12 days post inoculation. Tests on serum (n = 200) and oral fluid (n = 200) field samples from an ASFV-free population demonstrated that the assay was highly diagnostically specific. The convenience and diagnostic utility of oral fluid sampling combined with the flexibility to test either serum or oral fluid on the same platform suggests that this assay will be highly useful under the conditions for which OIE recommends ASFV antibody surveillance, i.e., in ASFV-endemic areas and for the detection of infections with ASFV isolates of low virulence.

Evaluation of oral mucosal transudate for immunodiagnosis of Chagas´ disease

Anticorpos anti-Trypanosoma cruzi (isotipo IgG) foram detectados no transudato da mucosa oral (TMO) através de um ensaio imunoenzimático. Foram estudados 21 indivíduos com doença de Chagas crônica comprovada através de diagnóstico clínico, eletrocardiográfico, epidemiológico e sorológico: 10 com forma cardíaca, 2 com forma digestiva, 6 com forma mista e 3 com forma assintomática. Sete indivíduos de área endêmica, com sorologia negativa, constituiram o grupo controle. O soro destes grupos foi armazenado a -20 oC. A coleta de TMO de ambos os grupos foi realizada com o dispositivo OraSureâ seguindo orientação do fabricante (OraSureâ , Epitope Inc., Beaverton, OR, USA). As amostras de TMO foram diluídas (1:2) e testadas em duplicata através de um ensaio imunoenzimático da Abbott Laboratories para detectar anticorpos IgG contra doença de Chagas. Vinte dos vinte e um pacientes chagásicos apresentaram densidade óptica acima do limiar de reatividade e foram considerados positivos para doença de Chagas. Nenhuma das amostras provenientes de indivíduos soronegativos foi positiva. A sensibilidade e especificidade foram de 95% e 100%, respectivamente. Estes resultados indicam que TMO poderá ser utilizado como um fluido biológico alternativo para o diagnóstico da doença de Chagas. Nós estamos aumentando o número de indivíduos para validar estes resultados incluindo a análise comparativa entre amostras de TMO e soro.

Determinação de etanol e voláteis relacionados em sangue e fluido oral por microextração em fase sólida em headspace associada à cromatografia gasosa com detector de ionização em chama

The aim of this study was to validate a method for the determination of acethaldehyde, methanol, ethanol, acetone and isopropanol employing solid-phase microextraction associated to gas chromatography with flame ionization detection. The operational conditions of SPME were optimized by response surface analysis. The calibration curves for all compounds were linear with r² > 0.9973. Accuracy (89.1-109.0%), intra-assay precision (1.8-8.5%) and inter-assay precision (2.2-8.2%) were acceptable. The quantification limit was 50 µg/mL. The method was applied to the meaurement of ethanol in blood and oral fluid of a group of volunteers. Oral fluid ethanol concentrations were not directly correlated with blood concentrations.

Distribution of free and conjugated cannabinoids in human bile samples.

The metabolism of Δ(9)-tetrahydrocannabinol (THC) is relatively complex, and over 80 metabolites have been identified. However, much less is known about the formation and fate of cannabinoid conjugates. Bile excretion is known to be an important route for the elimination of phase II metabolites. A liquid chromatography-tandem mass spectrometry LC-MS/MS procedure for measuring cannabinoids in oral fluid was adapted, validated and applied to 10 bile samples. THC, 11-hydroxy-Δ(9)-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THCCOOH), cannabinol (CBN), cannabidiol (CBD), Δ(9)-tetrahydrocannabinolic acid A (THC-A), 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol glucuronide (THCCOOH-gluc) and Δ(9)-tetrahydrocannabinol glucuronide (THC-gluc) were determined following solid-phase extraction and LC-MS/MS. High concentrations of THCCOOH-gluc were found in bile samples (range: 139-21,275 ng/mL). Relatively high levels of THCCOOH (7.7-1548 ng/mL) and THC-gluc (38-1366 ng/mL) were also measured. THC-A, the plant precursor of THC, was the only cannabinoid that was not detected. These results show that biliary excretion is an important route of elimination for cannabinoids conjugates and that their enterohepatic recirculation is a significant factor to consider when analyzing blood elimination profiles of cannabinoids. Furthermore, we suggest that the bile is the matrix of choice for the screening of phase II cannabinoid metabolites.

Faut-il autoriser les femmes à boire ou à manger durant l'accouchement [Should we allow women to eat or drink during labor?].

Cet article présente les résultats de la revue systématique: Singata M, Tranmer J, Gyte GM. Restricting oral fluid and food intake during labour. Cochrane Database Syst Rev. 2010 Jan 20;(1):CD003930. PMID: 20091553.

Ganho no desempenho uterino da parturiente com ingestão de mel e repercussões no recém-nascido

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Workplace drug testing, different matrices different objectives

Drug testing is used by employers to detect drug use by employees or job candidates. It can identify recent use of alcohol, prescription drugs, and illicit drugs as a screening tool for potential health and safety and performance issues. Urine is the most commonly used sample for illicit drugs. It detects the use of a drug within the last few days and as such is evidence of recent use; but a positive test does not necessarily mean that the individual was impaired at the time of the test. Abstention from use for three days will often produce a negative test result. Analysis of hair provides a much longer window of detection, typically 1 to 3 months. Hence the likelihood of a falsely negative test using hair is very much less than with a urine test. Conversely, a negative hair test is a substantially stronger indicator of a non-drug user than a negative urine test. Oral fluid (saliva) is also easy to collect. Drugs remain in oral fluid for a similar time as in blood. The method is a good way of detecting current use and is more likely to reflect current impairment. It offers promise as a test in post-accident, for cause, and on-duty situations. Studies have shown that within the same industrial settings, hair testing can detect twice as many drug users as urine testing. Copyright (C) 2012 John Wiley & Sons, Ltd.

Detection and identification of 700 drugs by multi-target screening with a 3200 Q TRAP LC-MS/MS system and library searching

The multi-target screening method described in this work allows the simultaneous detection and identification of 700 drugs and metabolites in biological fluids using a hybrid triple-quadrupole linear ion trap mass spectrometer in a single analytical run. After standardization of the method, the retention times of 700 compounds were determined and transitions for each compound were selected by a "scheduled" survey MRM scan, followed by an information-dependent acquisition using the sensitive enhanced product ion scan of a Q TRAP hybrid instrument. The identification of the compounds in the samples analyzed was accomplished by searching the tandem mass spectrometry (MS/MS) spectra against the library we developed, which contains electrospray ionization-MS/MS spectra of over 1,250 compounds. The multi-target screening method together with the library was included in a software program for routine screening and quantitation to achieve automated acquisition and library searching. With the help of this software application, the time for evaluation and interpretation of the results could be drastically reduced. This new multi-target screening method has been successfully applied for the analysis of postmortem and traffic offense samples as well as proficiency testing, and complements screening with immunoassays, gas chromatography-mass spectrometry, and liquid chromatography-diode-array detection. Other possible applications are analysis in clinical toxicology (for intoxication cases), in psychiatry (antidepressants and other psychoactive drugs), and in forensic toxicology (drugs and driving, workplace drug testing, oral fluid analysis, drug-facilitated sexual assault).

Pharmacokinetics and excretion of gamma-hydroxybutyrate (GHB) in healthy subjects

In Europe and the United States, the recreational use of gamma-hydroxy butyric acid (GHB) at dance clubs and "rave" parties has increased substantially. In addition, GHB is used to assist in the commission of sexual assaults. The aim of this controlled clinical study was to acquire pharmacokinetic profiles, detection times, and excretion rates in human subjects. Eight GHB-naïve volunteers were administered a single 25-mg/kg body weight oral dose of GHB, and plasma, urine, and oral fluid specimens were analyzed by using gas chromatography-mass spectrometry (GC-MS). Liquid-liquid extraction was performed after acid conversion of GHB to gamma-butyrolactone. Limits of quantitation of 0.1 (oral fluid), 0.2 (urine), and 0.5 microg/mL (plasma) could be achieved in the selected ion monitoring mode. GHB plasma peaks of 39.4 +/- 25.2 microg/mL (mean +/- SEM) occurred 20-45 min after administration. The terminal plasma elimination half-life was 30.4 +/- 2.45 min, the distribution volume 52.7 +/- 15.0 L, and the total clearance 1228 +/- 233 microL/min. In oral fluid, GHB could be detected up to 360 min, with peak concentrations of 203 +/- 92.4 microg/mL in the 10-min samples. In urine, 200 +/- 71.8 and 230 +/- 86.3 microg/mL, were the highest GHB levels measured at 30 and 60 min, respectively. Only 1.2 +/- 0.2% of the dose was excreted, resulting in a detection window of 720 min. Common side-effects were confusion, sleepiness, and dizziness; euphoria and change of vital functions were not observed. GHB is extensively metabolized and rapidly eliminated in urine and oral fluid. Consequently, samples should be collected as soon as possible after ingestion.