Identification of benzydamine and its metabolit in the presence of certain anti-inflammatory non-steroidal drugs

The aim of the work. Currently, a large number of cases of non-medical use of benzydamine hydrochloride have been described. The identification of benzydamine and its metabolite, benzydamine N-oxide, in the presence of some non-steroidal anti-inflammatory drugs, has been insufficiently studied. Therefore, the development of a method for its identification in biological material is an urgent task. Materials and methods. The subjects of the study were benzydamine hydrochloride and its metabolite, as well as some non-steroidal anti-inflammatory drugs, which are its analogues in terms of pharmacological action. The studies were carried out by methods of thin layer chromatography and high-performance liquid chromatography. Results. At the first stage a screening method for benzydamine identification was studied using the extraction in acidic and basic conditions. It was shown that benzydamine can be isolated in both medias with subsequent development with a solution of iodoplatinate and Dragendorff's reagent according to Munier or with Mandelin reagent respectively. The mobile phase was selected and respective hRf for the target molecule were defined. After a preliminary identification of benzydamine a reference method for the final confirmation of the drug that had led to poisoning was proposed. A robust, specific and accurate reversed phase HPLC method was chosen. It was shown that benzydamine exists in biological material mainly in a form of metabolite – benzydamine N-oxide. The selected method was able to separate and determine key analytes in biological samples after a preparative isolation by TLC method. The comparison with UV spectra of the reference standard of benzydamine hydrochloride was proposed to avoid false positive conclusion of drug identification. Conclusions. Proposed methodology can be applied for routine identification of benzydamine poisoning in toxicological laboratories

Ultra-fast direct method for identifying microorganisms from BACTEC lytic/10 anaerobic/F flasks

Background: Fast diagnosis of bloodstream infections remains the most important challenge for clinical microbiologists. The introduction of the mass-spectrometry represents a breakthrough, although several methods are already commonly used for the direct identification from positive blood cultures we present a faster method (ultra fast) for Lytic anaerobic flasks. Methods: We compare the ultra-fast (UF) method with the extensively employed differential centrifugation method (DC) and both to routine identification after 18–24 h of incubation. UF and DC method correlation rates to the gold standard were calculated, and statistical significance was proved with the Z test. Results: UF performed better overall than DC, with this difference being statistically significant. This tendency was observed in every subanalysis.

NGS-Based MRD Quantitation: An Alternative to qPCR Validated on a Large Consecutive Cohort of Children with ALL

Together with multicolor flow cytometry, quantitation of clonal immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements represents the current standard for the detection of minimal / measurable residual disease (MRD) in treatment protocols for pediatric acute lymphoblastic leukemia (ALL) patients. Despite the adoption of next generation sequencing (NGS) in the routine identification of clonal IG/TR gene rearrangements as markers for MRD detection, real-time quantitative (q)PCR is still the standard for MRD quantitation in follow-up samples. So far, no large-scale direct comparison of qPCR- and NGS-based MRD quantitation has been performed. We compared qPCR- and NGS-MRD evaluation in a cohort of children with B-cell precursor (BCP) ALL treated on the AIEOP-BFM ALL 2009 protocol and assessed the feasibility and relevance of this method for the stratification at day 33 (EOI). In total, 459 patients were diagnosed with BCP-ALL from 2010 to 2018, and 437 of them were included in our study based on the availability of residual DNA material isolated from day 33 bone marrow aspirates and having at least one IG/TR MRD marker detectable by standard qPCR with protocol-required sensitivity of 10 -4. Sequencing libraries were prepared according to the EuroClonality-NGS group SOP (Brüggemann et al, Leukemia 2019) with the total DNA input normalized to the equivalent of 150,000 nucleated cells to reach MRD sensitivity of 10 -5 and sequenced on Illumina NovaSeq and MiSeq instruments. In total of 780 IG/TR markers evaluated by both NGS and qPCR. Sequencing data were analyzed using the ARResT/Interrogate (Bystry et at, Bioinformatics 2017) pipeline and a custom bioinformatic analysis process and the NGS-MRD results were normalized to the EuroClonality-NGS central in-tube quality/quantification control (cIT-QC; Knecht et al, Leukemia 2019). From the total 780 IG/TR MRD markers evaluated by both methods, 629 (80.6%) were concordant with 242 markers being MRD positive and 387 negative. From 82 markers that were only positive by qPCR and not by NGS, 76 were positive below the quantitative range (positive non-quantifiable). Specificity analysis was performed for each marker by searching for the junction sequence across the dataset of all patients' NGS results. Based on these results, 22 out of 82 markers positive only by qPCR were classified as potentially unspecific (false positive) and similarly 32 unspecific markers were identified among the 69 positive only by NGS. This was also supported by unspecific amplification of the polyclonal control in 27 out of these 32 corresponding qPCR systems, in some cases leading to qPCR negative classification determined by the EuroMRD guidelines. Overall stratification of patients based only on day 33 MRD by qPCR or NGS was concordant in 76% of patients by both methods, while in 19% of patients, NGS-MRD quantitation led to the assignment to a lower-risk group, mainly due to the elimination of false-positive results. Furthermore, analysis of all positive markers across all patients' NGS libraries showed, that one out of 10 markers (mainly in the IGK, TRG and TRD loci) used for qPCR-MRD stratification did not provide satisfactory specificity, although they fully met EuroMRD criteria during the optimization of qPCR patient-specific assays. Our results show that NGS-MRD is highly concordant with traditional qPCR-based strategy and has comparable sensitivity and clinical value in the setting of a BFM-based clinical protocol, while being less laborious and providing significantly more specific results and additional information on the IG/TR repertoire (Kotrova et al, Blood 2016). Our study also emphasizes the importance of selecting MRD markers of adequate specificity at diagnosis. Currently, this selection can be assisted by these broad sequencing data on IG/TR repertoire of large number of patients. Based on these results, we propose that frontline NGS-MRD evaluation developed by the EuroClonality-NGS working group can be used as an alternative to traditional qPCR-based MRD quantitation in future MRD-based treatment protocols. Supported by grants NU20-03-00284 and NU20-07-00322 from the Czech Health Research Council and 534120 from Charles University. All methods were established through collaboration within the EuroClonality-NGS and EuroMRD groups. Disclosures van der Velden: Agilent: Research Funding; Navigate: Other: Service Level Agreement; Janssen: Other: Service Level Agreement; EuroFlow: Other: Service Level Agreement, Patents & Royalties: for network, not personally; BD Biosciences: Other: Service Level Agreement. Brüggemann: Incyte: Other: Advisory Board; Janssen: Speakers Bureau; Amgen: Other: Advisory Board, Travel support, Research Funding, Speakers Bureau. Langerak: Erasmus MS, University Medical Center: Current Employment; F. Hoffmann-La Roche Ltd/Genentech, Inc.: Research Funding; Gilead: Research Funding; Janssen: Speakers Bureau.

A NOTE ON VACCINIUM BRASSII SLEUMER (ERICACEAE), RECORDING FILAMENTOUS WAX ON THE FLOWERS

Routine identification of historical Vaccinium specimens at E revealed scurfy deposits on a specimen identified as Vaccinium brassii Sleumer. On examination with a Scanning Electron Microscope (SEM), these scurfy deposits turned out to be filamentous wax remarkably similar to that observed in Vaccinium ceraceum Argent. The disparity of the geographical locations of these two species is noted, together with the lack of molecular data to give insights into the evolutionary relationships.

Novel Method for Identifying Care Home Residents in England: A Validation Study

IntroductionThe ability to identify residents of care homes in routinely collected health care data is key to informing healthcare planning decisions and delivery initiatives targeting the older and frail population. Health-care planning and delivery implications at national level concerning this population subgroup have considerably and suddenly grown in urgency following the onset of the COVID-19 pandemic, which has especially hit care homes. The range of applicability of this information has widened with the increased availability in England of retrospectively collected administrative databases, holding rich patient-level details on health and prognostic status who have made or are in contact with the National Health Service. In practice lack of a national registry of care homes residents in England complicates assessing an individual's care home residency status, which has been typically identified via manual address matching from pseudonymised patient-level healthcare databases linked with publicly availably care home address information. ObjectivesTo examine a novel methodology based on linking unique care home address identifiers with primary care patient registration data, enabling routine identification of care home residents in health-care data. MethodsThis study benchmarks the proposed strategy against the manual address matching standard approach through a diagnostic assessment of a stratified random sample of care home post codes in England. ResultsDerived estimates of diagnostic performance, albeit showing a non-insignificant false negative rate (21.98%), highlight a remarkable true negative rate (99.69%) and positive predictive value (99.35%) as well as a satisfactory negative predictive value (88.25%). ConclusionsThe validation exercise lends confidence to the reliability of the novel address matching method as a viable and general alternative to manual address matching.

Epidemiology of clinically relevant Entamoeba spp. (E. histolytica/dispar/moshkovskii/bangladeshi): A cross sectional study from North India

Background Entamoeba infections have major impact on millions of the people worldwide. Entamoeba histolytica has long been accepted as the only pathogenic species. However, recent reports of other Entamoeba spp. in symptomatic cases have raised questions on their pathogenicity. Methodology/Principal findings Total 474 stool samples and 125 liver aspirates from patients with intestinal and extra intestinal manifestations and from community were included. Sewage samples from the hospital and the city were also included. Microscopic examination and molecular detection were performed to detect presence of E. histolytica/ dispar/ moshkovskii/ bangladeshi. The associated demographic and socioeconomic factors were statistically analyzed with the presence of Entamoeba. Microscopy detected Entamoeba spp. in 5.4% stool and 6.4% liver aspirate samples. Through nested multiplex PCR, prevalence of Entamoeba spp. in intestinal and extra-intestinal cases was 6.6% (20/301) and 86.4% (108/125) respectively and in asymptomatic population was 10.5% (13/123). Sewage samples did not show presence of any Entamoeba spp. Uneducated subjects, low economic conditions, untreated drinking water, consumption of raw vegetables and habit of not washing hands before meals were significantly associated with presence of Entamoeba spp. Conclusions E. histolytica still remains the only Entamoeba spp. in invasive extra intestinal infections. E. dispar was detected in both asymptomatic and symptomatic intestinal infections. Routine identification of Entamoeba spp. should incorporate PCR based detection methods.

Cydrasil 3, a curated 16S rRNA gene reference package and web app for cyanobacterial phylogenetic placement

Cyanobacteria are a widespread and important bacterial phylum, responsible for a significant portion of global carbon and nitrogen fixation. Unfortunately, reliable and accurate automated classification of cyanobacterial 16S rRNA gene sequences is muddled by conflicting systematic frameworks, inconsistent taxonomic definitions (including the phylum itself), and database errors. To address this, we introduce Cydrasil 3 (https://www.cydrasil.org), a curated 16S rRNA gene reference package, database, and web application designed to provide a full phylogenetic perspective for cyanobacterial systematics and routine identification. Cydrasil 3 contains over 1300 manually curated sequences longer than 1100 base pairs and can be used for phylogenetic placement or as a reference sequence set for de novo phylogenetic reconstructions. The web application (utilizing PaPaRA and EPA-ng) can place thousands of sequences into the reference tree and has detailed instructions on how to analyze results. While the Cydrasil web application offers no taxonomic assignments, it instead provides phylogenetic placement, as well as a searchable database with curation notes and metadata, and a mechanism for community feedback.

Direct Detection of Streptococcus suis from Cerebrospinal Fluid, Positive Hemoculture, and Simultaneous Differentiation of Serotypes 1, 1/2, 2, and 14 within Single Reaction

Streptococcus suis is an emerging zoonotic bacterium causing septicemia and meningitis in humans. Due to rapid disease progression, high mortality rate, and many underdiagnosed cases by time-consuming routine identification methods, alternative diagnostic testing is essential. Among 29 broadly accepted S. suis serotypes, serotypes 2 and 14 are high prevalent; however, many PCR assays showed an inability to differentiate serotype 2 from 1/2, and 1 from 14. In this study, we developed and validated a new multiplex PCR assay that facilitates the identification of only the 29 true serotypes of S. suis and simultaneously differentiates serotypes 1, 1/2, 2, and 14 within a single reaction. Importantly, the multiplex PCR could detect S. suis directly from positive hemocultures and CSF. The results revealed high sensitivity, specificity, and 100% accuracy with almost perfect agreement (κ = 1.0) compared to culture and serotyping methods. Direct detection enables a decrease in overall diagnosis time, rapid and efficient treatment, reduced fatality rates, and proficient disease control. This multiplex PCR offers a rapid, easy, and cost-effective method that can be applied in a routine laboratory. Furthermore, it is promising for developing point-of-care testing (POCT) for S. suis detection in the future.

Dipeptidyl Peptidase IV as a Novel Prognostic Marker and Important Therapeutic Target in Melanoma

BackgroundThere is a lot of evidence which suggests that DPP IV level may correlate with a type of tumor cells, metastatic potential and prognosis for the patient. Bearing in mind that the melanomas are characterized by high heterogeneity and identification of specific phenotypes of cells allows for early and more effective therapy, the aim of our study was to check whether there is a correlation between the DPPIV and the metastatic potential of melanoma cell lines. Additionally, the aim of our research was to evaluate the anti-tumor potential of linagliptin and saxagliptin in melanoma cell lines as well as determining correlation between cytotoxicity of the drugs and DPP IV level. MethodsThe inhibitory effect of tested drugs on the cancer cell growth was assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) while cell cycle analysis and apoptosis were performed using the NucleoCounter® NC-3000™ system (ChemoMetec, Denmark), following the instructions provided by the manufacturer. DPPIV release by cancer cells was measured by DPP4/CD26 ELISA assay kit for biological samples (Cloud-Clone Corp.,Wuhan,China). ResultsOur results showed that DPPIV overexpression promoted cell proliferation of melanoma cells. Our data showed that especially short term treatment with linagliptin is associated with not only decreased expression of DPPIV and inhibition of cell proliferation but also induction of cell cycle disruption and apoptosis in melanoma. ConclusionsThe routine identification of this glycoprotein in melanoma would be fundamental to assessing not only the risk of metastasis/disease progression but also selection of therapy and evaluation of its effectiveness.

The impact of vestibular symptoms and electronystagmography results on recovery from sudden sensorineural hearing loss

Objectives Idiopathic sudden sensorineural hearing loss (SSNHL) represents a frequently encountered otological entity, of various types and severity, with an array of associated symptoms including vertigo. This is a devastating life-changing condition with a blurry prognosis. The objective of this study was to determine the clinical association of vestibular impairment by electronystagmography (ENG) and caloric tests, and their ability to predict prognosis. Methods An observational, crossectional study was carried out amongst patients admitted with SSNHL. Each consenting patient had an audiometry test performed on admission as well as ENG and caloric tests. Treatment included oral steroids and carbogen with intratympanic steroids used only as salvage treatment. Follow-up was completed after 6 months when hearing gains were evaluated. Finally, an association was sought between the rate of recovery and ENG and caloric test results. Results Of 35 patients included, marked recovery was seen in patients without vertigo when compared to those with vertigo (p=0.003). A statistically significant association was found between the presence of vertigo and hearing deterioration (p=0.008). More so, normal electronystagmography results were associated with marked recovery (p=0.04). Conclusions The vestibular end organs are both subjectively and objectively affected in SSNHL as demonstrated by the abnormal ENG and caloric tests in our study despite the small sample size. Concomitant vestibular involvement carries poorer prognosis and routine identification may help foresee the recovery of patients with SSNHL and as such, aid in patient counseling. ENG and caloric tests are easily available and may be recommended for all patients with SSNHL.