Holistic study design following Quality by Design approach for fabrication of hybrid polymeric nanoparticulate based dry powders as carriers for Ciprofloxacin.

Resistance to antibiotics such as Ciprofloxacin (CIP) is becoming a critical issue and needs to be addressed globally. CIP is widely used because of manifold uses; however, the long-term therapy poses serious health risks including FDA black box warnings such as tendinitis and peripheral neuropathy. Therefore, nanotechnology-based products can be an effective measure to improve therapeutic outcomes by maintaining the dose at the target site while reducing the dose-dependent toxicity. Biodegradable and biocompatible polymers, Chitosan (CS) and Hyaluronic acid (HA) were used in this work due to their diverse biological characteristics. A simple yet economical ionic gelation method was employed to synthesize nanoparticles with a plexus-like network; nanoplexes, followed by spray-drying to obtain the dry powders to improve stability. Quality by Design (QbD) approach was utilized during the study for robustness and standardization followed by Design of Experiment (DoE) for optimization in a holistic way. The mean particle size of the optimized powder sample was found to be 301.1 nm with a percentage encapsulation efficiency (% EE) of 78.8 %. In-vitro dissolution studies corroborated the controlled release of CIP over 48 h. Also, mathematical kinetic modeling was applied to obtain thorough insight into the mechanism of drug release. Moreover, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were presented to be lower in the case of prepared dry powder as compared to CIP, stating that nanotechnology can improve antimicrobial activity.

When it rains it pours: an increased prevalence of intestinal carriage of vancomycin-resistant Enterococcus faecium related to higher use of oral vancomycin in a tertiary care Hungarian clinical center during the years of the COVID-19 pandemic.

OBJECTIVES: This study aims to investigate the association between oral vancomycin consumption and intestinal vancomycin-resistant Enterococcus carriage in the pre- and COVID era in the clinical center of the University of Szeged, Hungary. METHODS: This retrospective microbiological examination was carried out using electronically collected data, corresponding to the period between 1 January 2018 and 31 December 2022, at the Department of Medical Microbiology. Data included isolated species and the according antimicrobial susceptibility patterns. Annual consumption data for oral vancomycin consumption were exported from the database of the Central Pharmacy of the Clinical Center. As a strain typing typing procedure, Fourier transform infrared spectroscopy analysis was used. RESULTS: There was a significant increase in the number of fecal vancomycin-resistant Enterococcus isolates throughout the study. The prevalence increased significantly during the years of the pandemic. The use of orally administered vancomycin in the clinical center increased significantly. A strong positive correlation existed between the two phenomena. Several strains with different resistance patterns spread in the clinical center. Two of these occurred in greater numbers, differing in their high-level aminoglycoside resistance. However, the overall resistance of these strains was stagnating. FTIR analysis revealed that 59 of the 62 strains were also divided into two large clusters differing partially in their high-level aminoglycoside resistance. CONCLUSION: During the pandemic, intestinal VRE carriage among clinical center patients increased significantly, linked to increased oral vancomycin use. Different strains spread, with aminoglycoside resistance being the primary distinction. This highlights the negative impact of the pandemic on VRE carriage.

Decreased Expression of Placental Proteins in Recurrent Pregnancy Loss: Functional Relevance and Diagnostic Value.

Miscarriages affect 50-70% of all conceptions and 15-20% of clinically recognized pregnancies. Recurrent pregnancy loss (RPL, ≥2 miscarriages) affects 1-5% of recognized pregnancies. Nevertheless, our knowledge about the etiologies and pathophysiology of RPL is incomplete, and thus, reliable diagnostic/preventive tools are not yet available. Here, we aimed to define the diagnostic value of three placental proteins for RPL: human chorionic gonadotropin free beta-subunit (free-ß-hCG), pregnancy-associated plasma protein-A (PAPP-A), and placental growth factor (PlGF). Blood samples were collected from women with RPL (n = 14) and controls undergoing elective termination of pregnancy (n = 30) at the time of surgery. Maternal serum protein concentrations were measured by BRAHMS KRYPTOR Analyzer. Daily multiple of median (dMoM) values were calculated for gestational age-specific normalization. To obtain classifiers, logistic regression analysis was performed, and ROC curves were calculated. There were differences in changes of maternal serum protein concentrations with advancing healthy gestation. Between 6 and 13 weeks, women with RPL had lower concentrations and dMoMs of free ß-hCG, PAPP-A, and PlGF than controls. PAPP-A dMoM had the best discriminative properties (AUC = 0.880). Between 9 and 13 weeks, discriminative properties of all protein dMoMs were excellent (free ß-hCG: AUC = 0.975; PAPP-A: AUC = 0.998; PlGF: AUC = 0.924). In conclusion, free-ß-hCG and PAPP-A are valuable biomarkers for RPL, especially between 9 and 13 weeks. Their decreased concentrations indicate the deterioration of placental functions, while lower PlGF levels indicate problems with placental angiogenesis after 9 weeks.

Novel and rare ß-lactamase genes of Bacteroides fragilis group species: Detection of the genes and characterization of their genetic backgrounds.

OBJECTIVES: This study screened the prevalence of rare ß-lactamase genes in Bacteroides fragilis group strains from clinical specimens and normal microbiota and examined the genetic properties of the strains carrying these genes. METHODS: blaHGD1, blaOXA347, cblA, crxA, and pbbA were detected by real-time polymerase chain reaction in collections of Bacteroides strains from clinical (n = 406) and fecal (n = 184) samples. To examine the genetic backgrounds of the samples, end-point PCR, FT-IR, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were used. RESULTS: All B. uniformis isolates were positive for cblA in both collections. Although crxA was B. xylanisolvens-specific and associated with carbapenem resistance, it was only found in six fecal and three clinical B. xylanisolvens strains. Moreover, the crxA-positive strains were not clonal among B. xylanisolvens (contrary to cfiA in B. fragilis), implicating a rate of mobility or emergence by independent evolutionary events. The Phocaeicola (B.) vulgatus/P. dorei-specific gene blaHGD1 was detected among all P. vulgatus/P. dorei isolates from fecal (n = 36) and clinical (n = 26) samples. No blaOXA347-carrying isolate was found from European collections, but all US samples (n = 6) were positive. For three clinical isolates belonging to B. thetaiotaomicron (n = 2) and B. ovatus (n = 1), pbbA was detected on mobile genetic elements, and pbbA-positive strains displayed non-susceptibility to piperacillin or piperacillin/tazobactam phenotypically. CONCLUSIONS: Based on these observations, ß-lactamases produced by rare ß-lactamase genes in B. fragilis group strains should not be overlooked because they could encode important resistance phenotypes.

Seroprevalence of emerging hepatitis E virus in patients with acute hepatitis between 2004 and 2018 in Csongrád County, Hungary.

OBJECTIVES: Hepatitis E virus (HEV) has recently become endemic in Europe, however, it is often a remnant neglected by clinicians as the causative agent of acute and chronic hepatitis and is often misdiagnosed as a drug-induced liver injury. The infection rate in European pig farms is estimated to be around 15-20%, therefore, the primary source of HEV infections might be poorly prepared pork meat. As HEV infections may occur more often in clinical practice than previously thought, the present paper aims to analyse the seroprevalence of HEV in patients with acute hepatitis over a period of 14 years in Csongrád County, Hungary. METHODS: The sera of 4,270 hepatitis patients collected between 2004-2018 were tested for cumulative anti-HEV IgG/IgM. Furthermore, 170 IgM positive sera were tested for the presence of viral RNA by RT-qPCR. RESULTS: Between 2012-2018, the cumulative seroprevalence has increased 9.18 times, and between 2013-2018, IgM prevalence has increased 12.49 times. Viral RNA was detectable in 12.35% of IgM positive sera. CONCLUSION: The present paper presents data showing that the seroprevalence of hepatitis E virus has increased markedly over the course of the last decade in Hungary and in other European countries as well. The exact reason behind this phenomenon is yet to be determined. To assess the dynamics and the reason for this increase in prevalence, pan-European, multicentre studies should be conducted.

Quality by design-based optimization of in situ ionic-sensitive gels of amoxicillin-loaded bovine serum albumin nanoparticles for enhanced local nasal delivery.

A recommended first-line acute bacterial rhinosinusitis (ABR) treatment regimen includes a high dose of orally administered amoxicillin, despite its frequent systemic adverse reactions coupled with poor oral bioavailability. Therefore, to overcome these issues, nasal administration of amoxicillin might become a potential approach for treating ABR locally. The present study aimed to develop a suitable carrier system for improved local nasal delivery of amoxicillin employing the combination of albumin nanoparticles and gellan gum, an ionic-sensitive polymer, under the Quality by Design methodology framework. The application of albumin nanocarrier for local nasal antibiotic therapy means a novel approach by hindering the nasal absorption of the drug through embedding into an in situ gelling matrix, further prolonging the drug release in the nasal cavity. The developed formulations were characterized, including mucoadhesive properties, in vitro drug release and antibacterial activities. Based on the results, 0.3 % w/v gellan gum concentration was selected as the optimal in situ gelling matrix. Essentially, each formulation adequately inhibited the growth of five common nasal pathogens in ABR. In conclusion, the preparation of albumin-based nanoparticles integrated with in situ ionic-sensitive polymer provides promising ability as nanocarrier systems for delivering amoxicillin intranasally for local antibiotic therapy.

Risk Estimation of Gestational Diabetes Mellitus in the First Trimester.

CONTEXT: There is no early, first-trimester risk estimation available to predict later (gestational week 24-28) gestational diabetes mellitus (GDM); however, it would be beneficial to start an early treatment to prevent the development of complications. OBJECTIVE: We aimed to identify early, first-trimester prediction markers for GDM. METHODS: The present case-control study is based on the study cohort of a Hungarian biobank containing biological samples and follow-up data from 2545 pregnant women. Oxidative-nitrative stress-related parameters, steroid hormone, and metabolite levels were measured in the serum/plasma samples collected at the end of the first trimester from 55 randomly selected control and 55 women who developed GDM later. RESULTS: Pregnant women who developed GDM later during the pregnancy were older and had higher body mass index. The following parameters showed higher concentration in their serum/plasma samples: fructosamine, total antioxidant capacity, testosterone, cortisone, 21-deoxycortisol; soluble urokinase plasminogen activator receptor, dehydroepiandrosterone sulfate, dihydrotestosterone, cortisol, and 11-deoxycorticosterone levels were lower. Analyzing these variables using a forward stepwise multivariate logistic regression model, we established a GDM prediction model with a specificity of 96.6% and sensitivity of 97.5% (included variables: fructosamine, cortisol, cortisone, 11-deoxycorticosterone, SuPAR). CONCLUSION: Based on these measurements, we accurately predict the development of later-onset GDM (24th-28th weeks of pregnancy). Early risk estimation provides the opportunity for targeted prevention and the timely treatment of GDM. Prevention and slowing the progression of GDM result in a lower lifelong metabolic risk for both mother and offspring.

Prescription of Rifampicin for Staphylococcus aureus Infections Increased the Incidence of Corynebacterium striatum with Decreased Susceptibility to Rifampicin in a Hungarian Clinical Center.

Several reports have suggested a role for Corynebacterium striatum as an opportunistic pathogen. The authors have conducted a retrospective study at the Clinical Center of the University of Szeged, Hungary, between 2012 and 2021 that revealed significantly increased rifampicin resistance in this species. This work aimed to investigate the reasons behind this phenomenon. The data were collected corresponding to the period between 1 January 2012 and 31 December 2021 at the Department of Medical Microbiology, University of Szeged. To characterize the resistance trends, the antibiotic resistance index was calculated for each antibiotic in use. Fourteen strains with different resistance patterns were further analyzed with Fourier-transform infrared spectroscopy using the IR Biotyper®. The decline in C. striatum sensitivity to rifampicin seen during the COVID-19 pandemic may have been attributable to the use of Rifadin® to treat concomitant Staphylococcus aureus infections. The fact that the IR Biotyper® typing method revealed that the rifampicin-resistant C. striatum strains were closely related supports this hypothesis. The IR Biotyper® infrared spectroscopy proved to be a modern and fast method to support effective antimicrobial stewardship programs.

Development of Thermoresponsive-Gel-Matrix-Embedded Amoxicillin Trihydrate-Loaded Bovine Serum Albumin Nanoparticles for Local Intranasal Therapy.

A high dose of amoxicillin is recommended as the first-line therapy for acute bacterial rhinosinusitis (ABR). However, oral administration of amoxicillin is connected to many adverse reactions coupled with moderate bioavailability (~60%). Therefore, this study aimed to develop a topical nasal preparation of amoxicillin, employing a thermoresponsive nanogel system to increase nasal residence time and prolong drug release. Rheological investigations revealed that formulations containing 21−23% w/w Poloxamer 407 (P407) were in accordance with the requirement of nasal administration (gelling temperature ~35 °C). The average hydrodynamic diameter (<200 nm), pH (6.7−6.9), and hypertonic osmolality (611−663 mOsmol/L) of the in situ gelling nasal nanogel appeared as suitable characteristics for local rhinosinusitis treatment. Moreover, taking into account the mucoadhesive strength and drug release studies, the 21% w/w P407 could be considered as an optimized concentration for effective nasal delivery. Antibacterial activity studies showed that the ability of amoxicillin-loaded in situ gelling nasal nanogel to inhibit bacterial growth (five common ABR pathogens) preserved its effectiveness in comparison to 1 mg/mL amoxicillin aqueous solution as a positive control. Altogether, the developed amoxicillin-loaded in situ gelling thermoresponsive nasal nanogel can be a potential candidate for local antibiotic therapy in the nasal cavity.

Assessment of malformations, variations and diameters of vessels forming the circle of Willis - An autopsy study in a non-cerebrovascular cohort.

Background a purpose: The collateral capacity of the circle of Willis (CoW) may play an important role in the development of ischemic strokes. The occurrence of classical polygon shows wide geographical variations and morphological data on diameters of the Willisian collaterals are scarce. We aimed to assess CoW variations and vessel diameters in a Central European cohort. Subjects and methods: CoWs were removed during routine autopsy. The morphological pattern of the circles was recorded. The prepared circles were then put between two glass plates and tightly compressed. The length of the vessels and half of the circumference were measured under a light microscope enabling measurement with an approximation of 0.1 mm. Vessel diameters were calculated from vessel circumference. Results: A total of 110 circles were analysed. Incomplete circles (missing one or two segments of CoW) were found in 25 cases (22.7%). Any forms of anatomical variations were detected in 14 cases (12.7%). When applying the <1 mm diameter threshold for analysis, 36 anterior communicating arteries (32.7%), 53 right posterior communicating arteries (48.2%), 73 left posterior communicating arteries (66.4%) and 18 posterior communicating arteries (16.3%) on both the sides were considered hypoplastic. Conclusions: In patients without stroke in their history, complete CoW may be present in >60% of the cases. Our diameter data may serve as reference values for the Central-European population.

Erythromycin-loaded polymeric micelles: In situ gel development, in vitro and ex vivo ocular investigations.

Our present series of experiments was to create a value-added formulation that has the potential to exert a powerful and long-lasting antibacterial effect for use in ophthalmology. Erythromycin-loaded polymeric micelles were formulated with a micelle size of 87.14 nm in a monodisperse distribution with 86.94 % encapsulation efficiency. To decrease the polymeric micelle-like burst effect of these nanoparticles, the formulation was dispersed in a Carbopol 934P gel base to prolong the drug release and permeation profile of erythromycin. With successful incorporation, a short gelling time with proper sol to gel transition was experienced in the form of transparent gels. The optimized formulation has high mucoadhesion which is a critical factor for prolonging residence time. With the initial burst, the drug release was saturated with more than 75 % of the drug released in simulated tear fluid. Corneal permeability investigations revealed that the gel formulation provides the value-added properties of polymeric micelles, with elevated permeability through into the aqueous humour across the cornea. While retaining its antimicrobial activity, the formulation may be capable to be utilized as an innovative ophthalmic formulation for treating bacterial infections of the eye.

Machine learning-based typing of Salmonella enterica O-serogroups by the Fourier-Transform Infrared (FTIR) Spectroscopy-based IR Biotyper system.

BACKGROUND: Salmonella enterica is among the major burdens for public health at global level. Typing of salmonellae below the species level is fundamental for different purposes, but traditional methods are expensive, technically demanding, and time-consuming, and therefore limited to reference centers. Fourier transform infrared (FTIR) spectroscopy is an alternative method for bacterial typing, successfully applied for classification at different infra-species levels. AIM: This study aimed to address the challenge of subtyping Salmonella enterica at O-serogroup level by using FTIR spectroscopy. We applied machine learning to develop a novel approach for S. enterica typing, using the FTIR-based IR Biotyper® system (IRBT; Bruker Daltonics GmbH & Co. KG, Germany). We investigated a multicentric collection of isolates, and we compared the novel approach with classical serotyping-based and molecular methods. METHODS: A total of 958 well characterized Salmonella isolates (25 serogroups, 138 serovars), collected in 11 different centers (in Europe and Japan), from clinical, environmental and food samples were included in this study and analyzed by IRBT. Infrared absorption spectra were acquired from water-ethanol bacterial suspensions, from culture isolates grown on seven different agar media. In the first part of the study, the discriminatory potential of the IRBT system was evaluated by comparison with reference typing method/s. In the second part of the study, the artificial intelligence capabilities of the IRBT software were applied to develop a classifier for Salmonella isolates at serogroup level. Different machine learning algorithms were investigated (artificial neural networks and support vector machine). A subset of 88 pre-characterized isolates (corresponding to 25 serogroups and 53 serovars) were included in the training set. The remaining 870 samples were used as validation set. The classifiers were evaluated in terms of accuracy, error rate and failed classification rate. RESULTS: The classifier that provided the highest accuracy in the cross-validation was selected to be tested with four external testing sets. Considering all the testing sites, accuracy ranged from 97.0% to 99.2% for non-selective media, and from 94.7% to 96.4% for selective media. CONCLUSIONS: The IRBT system proved to be a very promising, user-friendly, and cost-effective tool for Salmonella typing at serogroup level. The application of machine learning algorithms proved to enable a novel approach for typing, which relies on automated analysis and result interpretation, and it is therefore free of potential human biases. The system demonstrated a high robustness and adaptability to routine workflows, without the need of highly trained personnel, and proving to be suitable to be applied with isolates grown on different agar media, both selective and unselective. Further tests with currently circulating clinical, food and environmental isolates would be necessary before implementing it as a potentially stand-alone standard method for routine use.

Corynebacterium striatum-Got Worse by a Pandemic?

The role of Corynebacterium striatum has been demonstrated in different nosocomial infections. An increasing number of publications have demonstrated its virulence in the respiratory tract, especially in the immunosuppressed patient population. The number of these patients has increased significantly during the COVID-19 pandemic. For this reason, we aimed to investigate the prevalence and antimicrobial resistance pattern of this species between 2012 and 2021 at the Clinical Center of the University of Szeged, Hungary. Altogether, 498 positive samples were included from 312 patients during the study period. On the isolates, 4529 antibiotic susceptibility tests were performed. Our data revealed that the prevalence of C. striatum increased during the COVID-19 pandemic, the rise occurred in respiratory, blood culture, and superficial samples. During the study period, the rifampicin resistance significantly increased, but others have also changed dynamically, including linezolid. The species occurred with diverse and changing co-pathogens in the COVID-19 era. However, the increasing rifampicin and linezolid resistance of C. striatum was probably not due to the most commonly isolated co-pathogens. Based on resistance predictions, vancomycin is likely to remain the only effective agent currently in use by 2030.

Phenotypic and Molecular Characterization of Carbapenem-Heteroresistant Bacteroides fragilis Strains.

Carbapenem-resistant Bacteroides fragilis strains usually emerge by an insertion sequence (IS) jump into the upstream region of the cfiA carbapenemase gene. However, intermediate or fully resistant cfiA-positive strains also exist. These do not have such IS element activations, but usually have heterogeneous resistance (HR) phenotypes, as detected by a disc diffusion or gradient tests. Heteroresistance is a serious antibiotic resistance problem, whose molecular mechanisms are not fully understood. We aim to characterize HR and investigate diagnostic issues in the set of cfiA-positive B. fragilis strains using phenotypic and molecular methods. Of the phenotypic methods used, the population analysis profile (PAP) and area under curve (AUC) measurements were the best prognostic markers for HR. PAP AUC, imipenem agar dilution and imipenemase production corresponded well with each other. We also identified a saturation curve parameter (quasi-PAP curves), which correlated well with these phenotypic traits, implying that HR is a stochastic process. The genes, on a previously defined 'cfiA element', act in a complex manner to produce the HR phenotype, including a lysine-acetylating toxin and a lysine-rich peptide. Furthermore, imipenem HR is triggered by imipenem. The two parameters that most correlate with the others are imipenemase production and 'GNAT' expression, which prompted us to suspect that carbapenem heteroresistance of the B. fragilis strains is stochastically regulated and is mediated by the altered imipenemase production.

Early pathways, biomarkers, and four distinct molecular subclasses of preeclampsia: The intersection of clinical, pathological, and high-dimensional biology studies.

Preeclampsia is a syndromic disease of the mother, fetus, and placenta. The main limitation in early and accurate diagnosis of preeclampsia is rooted in the heterogeneity of this syndrome as reflected by diverse molecular pathways, symptoms, and clinical outcomes. Gaps in our knowledge preclude successful early diagnosis, personalized treatment, and prevention. The advent of "omics" technologies and systems biology approaches addresses this problem by identifying the molecular pathways associated with the underlying mechanisms and clinical phenotypes of preeclampsia. Here, we provide a brief overview on how the field has progressed, focusing on studies utilizing state-of-the-art transcriptomics and proteomics methods. Moreover, we summarize our systems biology studies involving maternal blood proteomics and placental transcriptomics, which identified early maternal and placental disease pathways and showed that their interaction influences the clinical presentation of preeclampsia. We also present an analysis of maternal blood proteomics data which revealed distinct molecular subclasses of preeclampsia and their molecular mechanisms. Maternal and placental disease pathways behind these subclasses are similar to those recently reported in studies on the placental transcriptome. These findings may promote the development of novel diagnostic tools for the distinct subtypes of preeclampsia syndrome, enabling early detection and personalized follow-up and tailored care of patients.

Changes in resistance pattern of ESKAPE pathogens between 2010 and 2020 in the clinical center of University of Szeged, Hungary.

The acronym ESKAPE stands for six antibiotic-resistant bacterial pathogens namely, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. Monitoring their resistance is an important task for clinical microbiology laboratories. Our aim was to analyze the resistance patterns of these bacteria over ten years in clinical samples of our department. We examined the sample types from which these pathogens were most frequently isolated. The incidence of tests with resistant results for each pathogen in aggregate and the most important subgroups of each was also analyzed. We have also intended to predict the local priorities amongst these pathogens. The results of 1,268,126 antibiotic susceptibility tests performed on a total of 70,099 isolates over this period were examined. Most strains were derived from urine, blood culture, trachea, vagina, wounds, and abscesses. Prevalence of ESKAPE bacteria increased between 2011 and 2020 however, the steepest intensifications were seen in the cases of K. pneumoniae and P. aeruginosa. The number of antibiotic susceptibility tests with resistant results has also increased over the decade but the most notable increase was detected in E. faecium and A. baumannii. Based on the calculation of antimicrobial resistance index for each pathogen, the most serious challenges for us at present are A. baumannii, P. aeruginosa, and E. faecium and their multi-resistant forms. The theoretical prediction of proportion of resistant tests between 2020 and 2030 in our care area draws attention to a worrying trend in the cases of vancomycin-resistant E. faecium and carbapenem-resistant A. baumannii strains.

Comparison of four PCR and two point of care assays used in the laboratory detection of SARS-CoV-2.

Seeing the global emergence and the lack of a definitive cure for COVID-19, it is essential to find the most sensitive and specific detection method to identify infected patients in a timely manner. Our paper aims to compare the clinical sensitivity of different commercial RT-qPCR (Genesig, 1copy, DNA-Techonolgy and Charité primer-probe sets), isothermal PCR (Ustar Isothermal Amplification-Real Time Fluorescent Assay) and immunochromatographic antigen detection (BIOCREDIT COVID-19 Ag) assays developed to use in laboratory diagnosis of COVID-19. A total of 119 nasopharyngeal swab specimens were collected from symptomatic patients. A subset of samples, positive with two RT-qPCR assays were then tested with isothermal PCR and rapid antigen tests. Of the 119 specimens, 65 were positive by at least two PCR assays. All PCR assays showed substantial or perfect match, although some variations in the clinical performance was observed. Of the 37 and 32 remnant nasopharyngeal samples positive by RT-qPCR, respectively, three were positive by the BIOCREDIT COVID-19 Ag and 14 were detected by the isothermal amplification assay. In conclusion, in the clinical settings we recorded that each of the RT-qPCR assays was superior to other test formats, in particular, the routine use of the DNA-technology assay is recommended. Although alternative recommendations exist, we belive that the use of isothermal amplifiaction assays and antigen rapid tests for COVID-19 diagnosis can only serve as adjuncts while awaiting the PCR result because of their high false-negative rate.

Argonaute 2 Controls Antiviral Activity against Sweet Potato Mild Mottle Virus in Nicotiana benthamiana.

RNA silencing is a sequence specific post-transcriptional mechanism regulating important biological processes including antiviral defense in plants. Argonaute (AGO) proteins, the catalytic subunits of the silencing complexes, are loaded with small RNAs to execute the sequence specific RNA cleavage or translational inhibition. Plants encode several AGO proteins and a few of them, especially AGO1 and AGO2, have been shown to be required for antiviral silencing. Previously, we have shown that the P1 protein of the sweet potato mild mottle virus (SPMMV) suppresses the primary RNA silencing response by inhibiting AGO1. To analyze the role of AGO2 in antiviral defense against the SPMMV, we performed a comparative study using a wild type and ago2-/- mutant Nicotiana benthamiana. Here we show that the AGO2 of N. benthamiana attenuates the symptoms of SPMMV infection. Upon SPMMV infection the levels of AGO2 mRNA and protein are greatly increased. Moreover, we found that AGO2 proteins are loaded with SPMMV derived viral small RNAs as well as with miRNAs. Our results indicate that AGO2 protein takes over the place of AGO1 to confer antiviral silencing. Finally, we provide a plausible explanation for the AGO2 mediated recovery of an SPMMV-infected sweet potato.

Mining the red deer genome (CerEla1.0) to develop X-and Y-chromosome-linked STR markers.

Microsatellites are widely applied in population and forensic genetics, wildlife studies and parentage testing in animal breeding, among others, and recently, high-throughput sequencing technologies have greatly facilitated the identification of microsatellite markers. In this study the genomic data of Cervus elaphus (CerEla1.0) was exploited, in order to identify microsatellite loci along the red deer genome and for designing the cognate primers. The bioinformatics pipeline identified 982,433 microsatellite motifs genome-wide, assorted along the chromosomes, from which 45,711 loci mapped to the X- and 1096 to the Y-chromosome. Primers were successfully designed for 170,873 loci, and validated with an independently developed autosomal tetranucleotide STR set. Ten X- and five Y-chromosome-linked microsatellites were selected and tested by two multiplex PCR setups on genomic DNA samples of 123 red deer stags. The average number of alleles per locus was 3.3, and the average gene diversity value of the markers was 0.270. The overall observed and expected heterozygosities were 0.755 and 0.832, respectively. Polymorphic Information Content (PIC) ranged between 0.469 and 0.909 per locus with a mean value of 0.813. Using the X- and Y-chromosome linked markers 19 different Y-chromosome and 72 X-chromosome lines were identified. Both the X- and the Y-haplotypes split to two distinct clades each. The Y-chromosome clades correlated strongly with the geographic origin of the haplotypes of the samples. Segregation and admixture of subpopulations were demonstrated by the use of the combination of nine autosomal and 16 sex chromosomal STRs concerning southwestern and northeastern Hungary. In conclusion, the approach demonstrated here is a very efficient method for developing microsatellite markers for species with available genomic sequence data, as well as for their use in individual identifications and in population genetics studies.