Pharmaceutical residues have become micropollutants of emerging concern in wastewater. Insufficient removal by wastewater treatment plants (WWTPs) can result in negative ecotoxicological impacts on aquatic ecosystems. Therefore, the EU Urban Wastewater Treatment Directive (UWWTD 2024/3019) requires quaternary treatment upgrades and regular monitoring in European wastewater treatment plants. This necessitates efficient and easy-to-implement wastewater analysis methods. Consequently, this study presents a novel, sensitive, and reliable HRMS-based method for the simultaneous determination of twelve indicator substances (nine UWWTD-listed and three substitutes) in WWTP influent and effluent. The method is based on solid-phase extraction (SPE) of 2 mL wastewater samples, followed by reversed-phase ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) employing an Orbitrap Exploris™ 240. Detection was carried out using heated electrospray ionisation in positive ionisation mode (HESI+), except for diclofenac (HESI-). Quantification was performed using highly sensitive SIM scans, while high-resolution full-scan spectra were acquired for confirmation. Limits of quantification (LOQ) ranged mostly between 15 and 30 ng/L, except for candesartan, irbesartan, and diclofenac with LOQ > 50 ng/L. A laboratory validation demonstrated high precision and accuracy for all selected pharmaceuticals. Furthermore, the method was successfully applied to raw and treated wastewater from wastewater treatment plants (WWTPs) in the Saxon-Czech border region, which differ in terms of wastewater throughput and population equivalents. As a result, this method offers a rapid quantification approach for assessing the removal efficiency of pharmaceuticals and potential substitutes categorised in the UWWTD.

Objective: To evaluate adherence to and effects of standardized olfactory training (OT) after COVID-19 infection. Methods: In this prospective study, 475 patients (mean age 47.4/SD 9.5 years) with olfactory dysfunction (OD) after COVID-19 infection participated in OT on average 13 months (range 0-31 months) post-infection. Patients were assessed 3 times with an average interval of 3 months using questionnaires on quality of life (QoL), chemosensory functions, and OT adherence. Results: Overall, 363 patients (76.4%) took part in the first 2 and 49.1% in all 3 testing sessions. Before the initial assessment, 32.8% had performed OT in the past without any instructions, so that merely 9.1% adhered to recommended standards. By the second evaluation, after standardized OT was introduced to the participants, 31.1% (n = 106) reported following the training as recommended, decreasing to 18.5% (n = 43) by the third assessment, indicating that most participants did not complete the recommended duration of at least 12, preferably 24 to 36 weeks of olfactory training. QoL and subjective olfactory acuity significantly improved between the first and following testing sessions (P < .001), but these improvements were not associated with adherence to OT. However, male sex (P = .035), non-medical profession (P = .025), increased age (P = .009), and reduced QoL (P = .027) were observed factors for advanced training adherence to recommended standards. Conclusion: Overall, only a small proportion of participants consistently followed OT. Higher adherence, however, was associated with male sex, non-medical profession, older age, and greater impairment in QoL. Nevertheless, on average there was a clear improvement in QoL and subjective sense of smell over the testing period.

Epithelioid glioblastoma (eGB), a very aggressive and rare brain tumour, is associated with a dismal median overall survival. Effective therapies for patients with eGB, particularly with leptomeningeal dissemination, are still lacking. Here, we describe a case of a 25-year-old male diagnosed with an intramedullary cervical tumour with subsequent leptomeningeal disease. Histopathology identified a highly necrotising, epithelioid-type tumour with high cell density, most compatible with the diagnosis of an eGB. DNA analysis revealed an unprecedented B-Raf protooncogene, serine/threonine kinase (BRAF) gene variant in exon 15 (ENST00000288602.6, c.1799_1810delinsATG, p.(V600_W604delinsDG)), triggering activation of the mitogen-activated protein kinase (MAPK) pathway. Consequently, we initiated MAPK inhibitor (MAPKi) therapy, utilizing a combination of BRAF and mitogen-activated protein kinase kinase (MEK) inhibitors. Liquid chromatography–tandem mass spectrometry analysis confirmed the drugs’ presence in the patient’s cerebrospinal fluid, indicating their capacity to cross the blood-brain barrier. Remarkably, the patient responded very well to therapy and transitioned from a near-comatose state to significantly improved health, sustained for over three months. This study highlights that MAPKi, particularly targeted towards novel BRAFV600 mutations, might offer promising advancements in eGB treatment strategies.

BACKGROUND:Fortunately, the majority of COVID-19 patients recover from olfactory dysfunction (OD) within the first couple of weeks. However, from approximately 5% up to 20% continue to suffer from OD even more than 1 year after the onset. Nonetheless, factors associated with long-lasting OD are hardly known. The aim of this study was to identify favourable and disadvantageous markers of persisting OD in COVID-19 patients. METHODOLOGY:Sixty-six patients (46 female; mean age: 39.9 years) that suffer from OD longer than 6 months due to laboratory-confirmed SARS-CoV-2 infection have participated in this longitudinal study. Participants completed comprehensive psychophysical chemosensory tests (i.e., Sniffin' Sticks = TDI) and questionnaires twice at our department-on average 219 ± 80 (T-1) and 489 ± 89 (T-2) days after the onset of symptoms, respectively. Olfactory recovery rates were associated with demographic factors and questionnaires using linear regression analysis. RESULTS:Patients below 40 years of age improved better (TDI: 4.1 ± 4.3 vs. 0.7 ± 5.8; p = 0.008) and achieved statistically significant higher scores (TDI: 31.5 ± 4.0 vs. 27.3 ± 6.7; p = 0.033) regarding psychophysical chemosensory tests. Furthermore, linear regression analysis revealed that parosmia was associated with worse orthonasal smell function (T-1: β = -0.346, p = 0.004; T-2: β = -0.384, p = 0.001), especially concerning identification subtest (T-1: β = -0.395, p = 0.001; T-2: β = -0.398, p < 0.001). Moreover, increasing parosmia between T-1and T-2 led to worse orthonasal olfactory function (β = -0.294, p = 0.016). CONCLUSIONS:Older age and parosmia seem to be unfavourable factors of persisting OD in COVID-19 patients.

BACKGROUND:The direct thrombin inhibitor argatroban is indicated for the treatment of heparin-induced thrombocytopenia II, but it is also used off-label to treat critically ill patients presenting with heparin resistance, severe antithrombin deficiency, or hypercoagulability. Direct drug monitoring is not routinely available, and argatroban dosing is mainly based on global coagulation assays such as activated partial thromboplastin time (PTT) or diluted thrombin time (TT), both of which have limitations in patients with hypercoagulability. METHODS:Blood samples were obtained from critically ill patients treated with argatroban. Activated PTT and diluted TT were measured with a STA R Max3 analyzer (STAGO Deutschland GmbH, Germany) using an argatroban-calibrated kit. Ecarin clotting time was measured using a point-of-care viscoelastic test device. Liquid chromatography with tandem mass spectrometry was performed using a reversed-phase column, a solvent gradient, and an API4000 mass spectrometer with electrospray. Correlation was described using Pearson correlation coefficient r and Bayesian multilevel regression to estimate relationships between outcomes and covariates. RESULTS:From June 2021 to March 2022, 205 blood samples from 22 patients were analyzed, allowing for 195 activated PTT-liquid chromatography with tandem mass spectrometry comparisons, 153 ecarin clotting time-liquid chromatography with tandem mass spectrometry comparison, and 105 diluted TT-liquid chromatography with tandem mass spectrometry comparisons. Compared to liquid chromatography with tandem mass spectrometry, performance of argatroban quantification was best for diluted TT (r = 0.91), followed by ecarin clotting time (r = 0.58) and activated PTT (r = 0.48). Regression analysis revealed that patients with sepsis were more prone to argatroban overdosing (coefficient, 4.194; 95% credible interval, 2.220 to 6.792). CONCLUSIONS:Although activated PTT monitoring of argatroban is the most commonly used test, in critically ill patients, diluted TT provides more precise measurements. Alternately, point-of-care viscoelastic ecarin clotting time also provides guidance for argatroban dosing to identify overdosing if available. The data also suggested that patients with sepsis are at greater risk for argatroban overdosing. EDITOR’S PERSPECTIVE:

Radiation-induced morphea (RIM) is a rare complication of radiotherapy presenting as inflammatory fibrosis, most commonly reported in breast cancer patients. As underlying disease mechanisms are not well understood, targeted therapies are lacking. Since fibroblasts are the key mediators of all fibroproliferative diseases, this study aimed to characterize patient-derived fibroblasts to identify therapeutic targets. We studied primary human control and RIM-fibroblasts on a functional and molecular basis, analyzed peripheral blood and tissue samples and conducted, based on our findings, a treatment attempt in one patient. In RIM, we identified a distinct myofibroblast phenotype reflected by increased alpha-smooth-muscle-actin (αSMA) expression, reduced proliferation and migration rates, and overexpression of osteopontin (OPN). Our RNA sequencing identified aberrant Myc activation as a potential disease driver in RIM fibroblasts, similar to previous findings in systemic sclerosis. Treatment with the anti-inflammatory drug mesalazine reversed the myofibroblast phenotype by targeting Myc. Based on these findings, a patient with RIM was successfully treated with mesalazine, resulting in reduced inflammation and pain and tissue softening, while serum OPN was halved. The present study provides a comprehensive characterization of RIM fibroblasts, suggests a disease-driving role for Myc, demonstrates promising antifibrotic effects of mesalazine and proposes OPN as a biomarker for RIM.

AbstractObjectivesThe aim of the present study was to follow the daily course of patients with olfactory dysfunction and healthy controls and to assess (i) how many times a day, (ii) at which time, and (iii) in which aspect of daily life participants are conscious about their sense of smell.MethodsIn this longitudinal study, 49 patients with smell loss and 30 healthy participants were enrolled. Olfactory function was assessed using the Sniffin’ Sticks. All participants received paper diaries designed for a 14‐day period, featuring 12 rows representing 12 daily hours and six columns for various daily life aspects. They were instructed to mark their awareness of smell by indicating the relevant row and column in the diary. Following the return of the diaries, a second olfactory test was conducted within the patient group.ResultsOn average, patients were consciously aware of their sense of smell around 8 times daily, while healthy participants noted it about 6.5 times a day. Both groups primarily focused on their sense of smell during activities related to “eating,” followed by considerations in “social life” and “personal hygiene.” Interestingly, distinct patterns emerged: patients peaked in awareness at 8 a.m. and 7 p.m., whereas healthy individuals showed peaks at 6 a.m., 12 p.m., and 7 p.m. Despite regular diary use, we observed no improvement in patients' olfactory function or related quality of life.ConclusionThe olfactory diary is a valuable tool unveiling individual smell awareness patterns in patients with smell loss, aiding in counseling and patient management.Level of Evidence4

Hintergrund In den letzten Jahrzehnten wurden in der Standardisierung von quantitativen Riechtests große Fortschritte gemacht, wohingegen qualitative Riechstörungen nur durch Befragung der Betroffenen erfasst wurden. Mit der Entwicklung des Sniffin’ Sticks Parosmie Tests (SSParoT) wurde diese Lücke geschlossen. Das Ziel der vorliegenden Untersuchung war die Erfassung der hedonischen Bewertung von Duftstoffen mittels Kurzversion des SSParoT bei Patienten mit quantitativen Riechstörungen und der Vergleich der Ergebnisse abhängig vom Schweregrad der Riechstörung.

The small molecule A-485 competitively inhibits the histone acetyltransferase domain of CBP (cyclic-adenosine monophosphate response element-binding protein)/p300. Apart from its antineoplastic activity, researchers are exploring its potential benefits in treating osteoporosis and its impact on energy metabolism. However, so far, only limited pharmacokinetic data are available, and the crucial determination of A-485 concentration in various biological materials with small sample volumes remains unpublished. A rapid and sensitive LC-tandem mass spectrometry method has been developed and validated to quantify A-485 in mouse serum and tissue. In this method, serum samples underwent precipitation with acetonitrile, while cell lysates were appropriately diluted. The determination of A-485 utilized a reversed-phase column with a mobile phase gradient, and detection was carried out in multiple reaction monitoring mode. The lower standard sample, with a concentration of 7.8 ng/mL, served as the lower limit of quantification, while the upper standard was established at 1000 ng/mL. A-485 concentrations were assessed in both serum samples and the lysate of all examined tissues, revealing swift metabolic clearance. The analytical method outlined here is deemed appropriate for subsequent studies. The ability to measure the active ingredient in various compartments facilitates the determination of accurate pharmacokinetic parameters. In the event of human use of A-485, the analysis method can be seamlessly transferred to human samples.

Introduction Argatroban is indicated for treatment of heparin-induced thrombocytopenia, but is also used in critical ill COVID-19 patients presenting with extensive thrombin overload. Direct drug monitoring is not available and argatroban dosing is mainly based on activated partial plasmin time (aPTT), which has limitations in hypercoagulable patients with increased FVIII [1] [2]. The aim of this study was to compare correlation of routine clotting tests (aPTT, ecarin clotting time [ECA-CT] and diluted thrombin time [dTT]) [3] to argatroban plasma levels measured by gold standard mass spectrometry (LC/MS/MS).

Objective Loss of olfactory function is clinically tested by means of validated smell tests. However, the assessment of qualitative disorders (e.g., parosmia) has been performed by questionnaires only. This gap has been closed recently by the development of the Sniffin' Sticks Parosmia Test (SSParoT). The aim of the present study was the assessment of hedonic ratings of odors using the short version of the SSParoT and to compare the results according to the severity of smell loss.

Analysis of illicit drugs, medicines, and pathogens in wastewater is a powerful tool for epidemiological studies to monitor public health trends. The aims of this study were to (i) assess spatial and temporal trends of population-normalized mass loads of illicit drugs and nicotine in raw wastewater in the time of regulations against SARS-CoV-2 infections (2020–21) and (ii) find substances that are feasible markers for characterizing the occurrence of selected drugs in wastewater. Raw sewage 24-h composite samples were collected in catchment areas of 15 wastewater treatment plants (WWTPs) in urban, small-town, and rural areas in Germany during different lockdown phases from April 2020 to December 2021. Parent substances (amphetamine, methamphetamine, MDMA, carbamazepine, gabapentin, and metoprolol) and the metabolites of cocaine (benzoylecgonine) and nicotine (cotinine) were measured. The daily discharge of WWTP influents were used to calculate the daily load (mg/day) normalized by population equivalents (PE) in drained catchment areas (in mg/1,000 persons/day). A weekend trend for illicit drugs was visible with higher amounts on Saturdays and Sundays in larger WWTPs. An influence of the regulations to reduce SARS-CoV-2 infections such as contact bans and border closures on drug consumption has been proven in some cases and refuted in several. In addition, metoprolol and cotinine were found to be suitable as marker substances for the characterization of wastewater. A change in drug use was visible at the beginning of the SARS-CoV-2 crisis. Thereafter from mid-2020, no obvious effect was detected with regard to the regulations against SARS-CoV-2 infections on concentration of drugs in wastewater. Wastewater-based epidemiology is suitable for showing changes in drug consumption during the COVID-19 lockdown.

BACKGROUND:Migraine shows a cyclic pattern with an inter-ictal-, a pre-ictal, an ictal- and a post-ictal phase. We aimed to examine changes in psychophysical parameters during the migraine cycle.METHODS:The perception of nociceptive and non-nociceptive stimuli and an electrically induced axon-reflex-erythema were assessed in 20 healthy controls and 14 migraine patients on five consecutive days according to different phases of the migraine cycle. Pain was rated three times during a 10-second electrical stimulus. The size of the axon-reflex-erythema was determined using laser-Doppler-imaging. Intensity and hedonic estimates of odours presented by Sniffin' Sticks were rated.RESULTS:In healthy controls, no significant changes over the test days were observed. In migraine patients pain thresholds at the head decreased with an ictal minimum. Less habituation after five seconds of stimulation at the head was found pre-ictally, whereas reduced habituation to 10-second electrical stimulation was present in all phases. The axon-reflex-erythema size showed an inter-ictal-specific minimum at the head. odours were perceived ictally as more unpleasant and intense.CONCLUSIONS:Somatosensory functions, pain thresholds and habituation as predominantly central parameters, axon-reflex-erythema as a peripheral function of trigeminal neurons and odour perception as a predominantly extra-thalamic sensation change specifically over the migraine cycle indicating complex variations of neuronal signal processing.

Dependent on the excretion pattern, wastewater monitoring of viruses can be a valuable approach to characterizing their circulation in the human population. Using polyethylene glycol precipitation and reverse transcription-quantitative PCR, the occurrence of RNA of SARS-CoV-2 and influenza viruses A/B in the raw wastewater of two treatment plants in Germany between January and May 2022 was investigated. Due to the relatively high incidence in both exposal areas (plant 1 and plant 2), SARS-CoV-2-specific RNA was determined in all 273 composite samples analyzed (concentration of E gene: 1.3 × 104 to 3.2 × 106 gc/L). Despite a nation-wide low number of confirmed infections, influenza virus A was demonstrated in 5.2% (concentration: 9.8 × 102 to 8.4 × 104 gc/L; plant 1) and in 41.6% (3.6 × 103 to 3.0 × 105 gc/L; plant 2) of samples. Influenza virus B was detected in 36.0% (7.2 × 102 to 8.5 × 106 gc/L; plant 1) and 57.7% (9.6 × 103 to 2.1 × 107 gc/L; plant 2) of wastewater samples. The results of the study demonstrate the frequent detection of two primary respiratory viruses in wastewater and offer the possibility to track the epidemiology of influenza by wastewater-based monitoring.