ABSTRACT
OBJECTIVES: Linezolid-resistant opportunistic human pathogens Enterococcus faecalis and Enterococcus faecium are emerging health threats as limited therapeutic options remain. The aim of this study was to investigate the epidemiology, resistance mechanisms, and genetic diversity of linezolid-resistant enterococci (LRE) isolated between 2013 and 2021 and received at the Belgian National Reference Centre (NRC) for Enterococci. METHODS: Linezolid susceptibility testing was performed upon request on 2458 submitted enterococci strains. Whole-genome sequencing was performed on all LRE strains. RESULTS: Seventy-eight LRE human isolates, of which 63 (81%) E. faecalis and 15 (19%) E. faecium strains, were submitted to the Belgian NRC for Enterococci. Of the linezolid-resistant E. faecalis strains, 97% harboured the optrA gene (56% wild-type pE349) and 3% the poxtA gene. Of the linezolid-resistant E. faecium strains, 54% harboured the G2576T point mutation in the V domain of the 23S rRNA genes, 23% the poxtA, and 23% the optrA gene. Furthermore, two E. faecium strains were identified with a combination of two resistance mechanisms ([i] optrA and poxtA, and [ii] cfr(B) and G2576T point mutation, respectively). Vancomycin resistance was observed in 15% (n = 12) of the LRE. ST480 (n = 42/63 typed strains, 67%) was the most frequently detected sequence type (ST) in linezolid-resistant E. faecalis strains, while ST203 (n = 5/15 typed strains, 33%) was the most frequently detected ST in linezolid-resistant E. faecium strains. CONCLUSIONS: E. faecalis isolates harbouring optrA were the predominant LRE in Belgium, with ST480 as the most prominent multilocus sequence typing. Linezolid resistance in E. faecium could be attributed to either chromosomal mutations or transferable resistance determinants.
ABSTRACT
Toxicological data on overdose with human immunodeficiency virus inhibitors are scarce. We present a case report of two independent suicide attempts by self-administered overdose with the same antiretroviral medicine Genvoya® (emtricitabine/elvitegravir/tenofovir alafenamide/cobicistat). Both patients were admitted to the hospital and presented with a loss of consciousness, lactic acidosis, elevated hepatic transaminase levels and hemodynamic instability. While one patient survived with advanced supportive measures, the other passed away. Emtricitabine levels were measured in vivo in various consecutive serum samples and postmortem urine, peripheral and cardiac serum samples and confirmed excessive use in both cases. This is the first time that emtricitabine levels following overdose are reported. Although measured concentrations for emtricitabine were quite similar in these cases, metabolic acidosis was more pronounced in the fatal case. The difference in outcomes between the two could be due to a difference in physiological status, susceptibility to accumulation and adverse effects, and perhaps a varying interval between ingestion and the start of supportive measures.
Subject(s)
Anti-HIV Agents , Drug Overdose , HIV Infections , Humans , Elvitegravir, Cobicistat, Emtricitabine, Tenofovir Disoproxil Fumarate Drug Combination/therapeutic use , Anti-HIV Agents/toxicity , Anti-HIV Agents/therapeutic use , HIV Infections/drug therapy , HIV , Drug Combinations , Emtricitabine/toxicity , Emtricitabine/therapeutic useABSTRACT
The use of saliva for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sparks debate due to presumed lower sensitivity and lack of standardization. Our aim was to evaluate the performance characteristics of (i) saliva collected by the ORAcollectTM device as a matrix for SARS-CoV-2 reverse-transcriptase polymerase chain reaction (RT-PCR), and (ii) 2 saliva rapid antigen tests (AgRDT). From 342 ambulatory individuals, both a nasopharyngeal swab and saliva sample via ORAcollectTM were obtained for a SARS-CoV-2 RT-PCR test. Furthermore, 54 and 123 additionally performed the V-ChekTM or WhistlingTM saliva AgRDT. In total, 35% of individuals screened positive for SARS-CoV-2 via nasopharyngeal swab. Saliva, as a matrix for the RT-PCR, had a specificity of 96.5% and a negative predictive value (NPV) of 91.3%. Interestingly, 6 out of 8 patients thought to be false positive in saliva re-tested positive by nasopharyngeal sampling after 2 to 9 days. Both V-ChekTM and WhistlingTM AgRDT had a lack of sensitivity, resulting in an NPV of 66.9 and 67.3%, respectively. Saliva proved to be a sensitive and specific matrix for SARS-CoV-2 detection by the RT-PCR. In this setting, saliva might have an earlier window of detection than the nasopharyngeal swab. By contrast, both AgRDT showed an unacceptably low sensitivity and NPV.
Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19 Testing , Clinical Laboratory Techniques/methods , Humans , Nasopharynx , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , Saliva , Sensitivity and Specificity , Specimen Handling/methodsABSTRACT
Obesity is a major public health problem. Gut microbiome dysbiosis has been associated with obesity, however, little is known on the effect of the dysbiosis on the microbiotic bio-transformation of xenobiotics. Fecal samples of lean (n = 9) and obese (n = 4) female volunteers were collected and characterized by 16 S rDNA gene sequencing. The microbiotic biotransformation of chlorogenic acid was studied using the collected fecal samples of lean and obese subjects in the colon stage of the gastro-intestinal dialysis model with colon stage (GIDM-Colon). The concentration of anaerobic bacteria was lower for all obese samples in comparison to the samples of the lean volunteers. Differences in gut microbiome composition and bacterial concentration were observed between the two populations. The obese gut microbiome presented a lower metabolic activity in comparison to the lean population. Chlorogenic acid was completely biotransformed after 24 h colonic dialysis in the lean population while it was still present in the obese population. Furthermore, 23 and 13 biotransformation products were identified in the lean and obese population, respectively; 11 unique biotransformation products from the caffeic, feruoylquinic and quinic acid pathways were identified in the lean population. The results confirm that changes in gut microbiota related to obesity are associated with differences in microbiotic biotransformation of xenobiotics and thus possibly influencing the activity, bioavailability and toxicity of orally administered xenobiotics and drugs.
Subject(s)
Chlorogenic Acid , Renal Dialysis , Biotransformation , Chlorogenic Acid/metabolism , Dysbiosis , Feces/microbiology , Female , Humans , ObesityABSTRACT
Spike (S)- and nucleocapsid (N)-specific serological assay responses were determined before and/or after first dose SARS-CoV-2 vaccination in 22 individuals. S-specific assays quantified antibodies after vaccination with significant higher levels in participants with a previous infection. Be cautious combining N-/S-specific assay results, potentially differentiating post-infection/vaccination immunization as assay-specific N-antibody waning was observed.
Subject(s)
Antibodies, Viral/blood , COVID-19 Vaccines/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Antibodies, Viral/immunology , COVID-19/diagnosis , COVID-19/prevention & control , COVID-19 Serological Testing , Coronavirus Nucleocapsid Proteins/immunology , Health Personnel , Humans , Phosphoproteins/immunology , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/immunology , VaccinationABSTRACT
SCOPE: Chlorogenic acid (5-caffeoylquinic acid), the most prominent polyphenolic compound in coffee, has been attributed multiple health-promoting effects such as anti-inflammatory, antidiabetic and antioxidative effects. These effects are dependent on the bioavailability of chlorogenic acid, which is determined by the pharmacokinetic properties: absorption, distribution, metabolism and excretion (ADME). In order to have a better understanding of the biological properties of chlorogenic acid and to optimize formulation and dosing of chlorogenic acid-containing food supplements, information on the absorption of chlorogenic acid and its microbial biotransformation products is of essence. METHODS AND RESULTS: In the present work, the intestinal absorption of chlorogenic acid and quinic acid, one of its most prominent intestinal biotransformation products, was studied by an in vitro permeability assay using a human Caco-2 cell line model. For both chlorogenic acid and quinic acid, the involvement of an active efflux mechanism was demonstrated, suggesting an overall low intestinal absorption. CONCLUSIONS: An overall low intestinal absorption for chlorogenic acid and quinic acid was reported given the involvement of an active efflux mechanism. These findings could aid in the development of optimal formulation and dosing strategies of chlorogenic acid in food supplements in order to obtain beneficial health effects.
Subject(s)
Chlorogenic Acid/metabolism , Intestinal Absorption/physiology , Quinic Acid/metabolism , Caco-2 Cells , Humans , In Vitro TechniquesABSTRACT
Recent data clearly show that the gut microbiota plays a significant role in the biotransformation of many endogenous molecules and xenobiotics, leading to a potential influence of this microbiotic metabolism on activation, inactivation and possible toxicity of these compounds. To study the colonic biotransformation of xenobiotics by the gut microbiome, in vitro models are often used as they allow dynamic and multiple sampling overtime. However, the pre-analytical phase should be carefully optimized to enable biotransformation product identification representative for the in vivo situation. During this study, chlorogenic acid was used as a model compound to optimize a ready-to-use gut microbiome biotransformation platform using an in vitro gastrointestinal dialysis-model with colon phase together with an instrumental platform using liquid chromatography coupled to high resolution mass spectrometry (LC-QTOF-MS). Identification of the biotransformation products of chlorogenic acid was performed using complementary suspect and non-targeted data analysis approaches (MZmineâ¯+â¯R and MPP workflow). Concerning the pre-analytical phase, (i) the influence of different incubation media (Wilkins-Chalgren Anaerobic Broth (WCB) and (versus) phosphate buffer) and different incubation times (prior to implementation in the colonic stage of the dialysis model) on fecal bacterial composition and concentration were investigated and (ii) four different sample preparation methods (centrifugation, extraction, sonication and freeze-drying) were evaluated targeting colonic biotransformation of chlorogenic acid. WCB as incubation medium showed to introduce substantial variation in the bacterial composition of the fecal samples, while the sterile phosphate buffer guaranteed a closer resemblance to the in vivo composition. Furthermore, incubation during 24â¯h in sterile phosphate buffer as medium showed no significant increase or decrease in anaerobic bacterial concentration, concluding that incubation prior to the colonic stage is not needed. Concerning sample preparation, centrifugation, sonication and extraction gave similar results, while freeze-drying appeared to be inferior. The extraction method was selected as an optimal sample preparation method given the quick execution together with a good instrumental sensitivity. This study optimized a ready-to-use platform to investigate colonic biotransformation of xenobiotics by using chlorogenic acid as a model compound. This platform can be used in the future to study differences in colonic biotransformation of xenobiotics using fecal samples of different patient groups.
Subject(s)
Bacteria/metabolism , Biotransformation/physiology , Chlorogenic Acid/metabolism , Gastrointestinal Microbiome/physiology , Gastrointestinal Tract/metabolism , Adult , Chromatography, Liquid/methods , Colon/metabolism , Feces/microbiology , Female , Humans , Mass Spectrometry/methods , Middle Aged , Renal Dialysis/methods , Xenobiotics/metabolismABSTRACT
The use of synthetic cannabinoids causes similar effects as Δ9 -tetrahydrocannabinol and long-term (ab)use can lead to health hazards and fatal intoxications. As most investigated synthetic cannabinoids undergo extensive biotransformation, almost no parent compound can be detected in urine, which hampers forensic investigations. Limited information about the biotransformation products of new synthetic cannabinoids makes the detection of these drugs in various biological matrices challenging. This study aimed to identify the main in vitro biotransformation pathways of 5Cl-THJ-018 and to compare these findings with an authentic urine sample of a 5Cl-THJ-018 user. The synthetic cannabinoid was incubated with pooled human liver microsomes and cytosol to simulate phase I and phase II biotransformations. Resulting extracts were analyzed with liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Three different data analysis workflows were applied to identify biotransformation products. A suspect screening workflow used an in-house database built from literature data and in silico biotransformation predictions. Two non-target screening workflows used a commercially available software and an open-source software for mass spectrometry data processing. A total of 23 in vitro biotransformation products were identified, with hydroxylation, oxidative dechlorination, and dihydrodiol formation pathways as the main phase I reactions. Additionally, five glucuronidated and three sulfated phase II conjugates were identified. The predominant in vivo pathway was through oxidative dechlorination and in total six metabolites of 5Cl-THJ-018 were identified. Biotransformation products both in vitro and in vivo were successfully identified using complementary suspect and non-target screening workflows.
Subject(s)
Cannabinoids/metabolism , Metabolic Detoxication, Phase II , Metabolic Detoxication, Phase I , Biotransformation , Cannabinoids/pharmacokinetics , Cannabinoids/urine , Chromatography, Liquid , Designer Drugs/metabolism , Designer Drugs/pharmacokinetics , Humans , Microsomes, Liver/metabolism , Middle Aged , Tandem Mass Spectrometry , WorkflowABSTRACT
Designer benzodiazepines have recently emerged as a class of new psychoactive substances. These substances are used in recreational settings and as alternatives to prescription benzodiazepines as self-medication for patients suffering from anxiety or other mental disorders. Due to the limited information available on the metabolic fate of these new substances, it is challenging to reliably detect their usage in bioanalytical (e.g. clinical and forensic) settings. The objective of this study was to investigate the in vitro Phase I and Phase II metabolism of the new designer benzodiazepine cloniprazepam and identify potential biomarkers for its detection in human biological fluids. Cloniprazepam was incubated with human liver microsomes and cytosolic fractions to generate both Phase I and II metabolites. The extracts were analysed using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. Identification of the metabolites was performed using two complementary workflows, including a suspect screening based on in silico predictions and a non-targeted screening. A total of nine metabolites were identified, eight Phase I metabolites and one Phase II metabolite, of which five were specific for cloniprazepam. Clonazepam was the major metabolite of cloniprazepam. Hydroxy-cloniprazepam, dihydroxy-cloniprazepam, 3-keto-cloniprazepam, 7-amino-cloniprazepam, hydroxy-clonazepam, 7-amino-clonazepam and 3-hydroxy-7-amino-clonazepam were formed through oxidation, hydroxylation, and/or reduction of the nitro-group. Glucuronidated hydroxy-cloniprazepam was the only Phase II metabolite detected. Five metabolites were specific for cloniprazepam. This study provided a set of human in vitro biotransformation products which can assist specific detection of cloniprazepam consumption in future studies.