Spa diversity and genetic characterization of t127 methicillin-resistant Staphylococcus aureus in a tertiary Greek hospital

Introduction: Methicillin-resistant Staphylococcus aureus (MRSA) causes severe community and hospital acquired infections. Identification of staphylococcal cassette chromosome mec (SCCmec), multilocus-sequence typing, and sequencing of S. aureus protein A (spa) gene are used for MRSA typing. The aim was to investigate the spa types of MRSA isolates in a tertiary hospital in Greece and analyse the whole genome sequences of two t127 MRSA isolates. Methods: Totally, 39 MRSA isolates collected from July 2019 to June 2020 in "Georgios Gennimatas" General Hospital of Thessaloniki, Greece, were included in the study. Identification and antimicrobial susceptibility testing were performed using VITEK II automated system, and spa typing was performed. A minimum spanning tree was used to display the spa type frequencies and the genetic distances among them. Two t127-MRSA isolates (IM-MRSA and PD-MRSA) were selected for WGS. Results: Six isolates (15.4%) were resistant to mupirocin, 18 (46.2%) to fusidic acid, three (7.7%) to vancomycin and two (5.1%) to teicoplanin. Twenty-two different spa types were detected, with t002, t003, and t422 being the most frequent (5/39, 12.8% each), followed by t1994 (4/39, 10.3%). The isolates presented high genetic diversity and, taking into account the time between hospital admission and sampling, intrahospital spread did not occur. Even the two t127 isolates were assigned to different sequence types, ST9-XII-t127 and ST1-IVa-t127. Plasmids and genes conferring antimicrobial resistance and virulence were also identified. Conclusions: Various spa types were identified and together with the information about the time between hospital admission and sampling supports polyclonal MRSA spread in the hospital excluding a nosocomial infection. WGS provides a more detailed analysis distinguishing even the isolates belonging to the same spa type

West Nile virus spread in Europe: phylogeographic pattern analysis and key drivers

BACKGROUND: West Nile virus (WNV) outbreaks in birds, humans, and livestock have occurred in multiple areas in Europe and have had a significant impact on animal and human health. The patterns of emergence and spread of WNV in Europe are very different from those in the US and understanding these are important for guiding preparedness activities.METHODS: We mapped the evolution and spread history of WNV in Europe by incorporating viral genome sequences and epidemiological data into phylodynamic models. Spatially explicit phylogeographic models were developed to explore the possible contribution of different drivers to viral dispersal direction and velocity. A "skygrid-GLM" approach was used to identify how changes in environments would predict viral genetic diversity variations over time.FINDINGS: Among the six lineages found in Europe, WNV-2a (a sub-lineage of WNV-2) has been predominant (accounting for 73% of all sequences obtained in Europe that have been shared in the public domain) and has spread to at least 14 countries. In the past two decades, WNV-2a has evolved into two major co-circulating clusters, both originating from Central Europe, but with distinct dynamic history and transmission patterns. WNV-2a spreads at a high dispersal velocity (88km/yr-215 km/yr) which is correlated to bird movements. Notably, amongst multiple drivers that could affect the spread of WNV, factors related to land use were found to strongly influence the spread of WNV. Specifically, the intensity of agricultural activities (defined by factors related to crops and livestock production, such as coverage of cropland, pasture, cultivated and managed vegetation, livestock density) were positively associated with both spread direction and velocity. In addition, WNV spread direction was associated with high coverage of wetlands and migratory bird flyways.CONCLUSION: Our results suggest that-in addition to ecological conditions favouring bird- and mosquito- presence-agricultural land use may be a significant driver of WNV emergence and spread. Our study also identified significant gaps in data and the need to strengthen virological surveillance in countries of Central Europe from where WNV outbreaks are likely seeded. Enhanced monitoring for early detection of further dispersal could be targeted to areas with high agricultural activities and habitats of migratory birds.</p

Investigation of the role of the ET, MTHFR and 5-HT gene polymorphisms in the pathogenesis of childhood migraine and functional abdominal pain

This study aims to investigate the common genetic substrate of migraine and functional abdominal pain in children and to correlate the genetic polymorphisms with pathogenesis, diagnosis and prognosis of those recurrent syndromes. The study includes 524 samples, 318 of which are patients (42 in Group A, 59 in Group B and 39 in Group C), 178 belong to the control group, and 206 samples are derived from patients’ families. For the investigation of a common genetic substrate of the two recurrent syndromes, 14 SNPs (ΕΤ1-Κ198Ν, ΕΤ1-138InsA, ΕΤΑ-231G/A, ETA-1363C/T, ETB-L277L, Ser-102T/C, Ser-1438A/G, MTHFR C677T, ΤΝFα-238 G/A, TNFα-308 G/A, TNFα-376G/A, TNFα-857C/T, TNFα-1031T/C and TNFB) were genotyped by polymerase chain reaction. Results from the statistical analysis suggest that: a) there is not a common genetic substrate between migraine and functional abdominal pain in children according to the 14 analyzed gene polymorphisms, b) there is a positive association of the MTHFR C677T gene polymorphism with migraine in group, B c) there is a positive association of the ETB-L277L and TNFB gene polymorphisms with functional abdominal pain (Group C), d) the haplotype [3Α]GGGGΤTA (gene polymorphisms located on chromosome 6- ΕΤ1-138InsA, ΕΤ1-Κ198Ν, ΤΝFα-238G/A, TNFα-308G/A, TNFα-376G/A, TNFα-857C/T, TNFα-1031T/C, ΤΝF-B) is an important risk/susceptibility haplotype and is positively associated with functional abdominal pain in Group C.Σκοπός της μελέτης ήταν η διερεύνηση κοινού γενετικού υποστρώματος μεταξύ της ημικρανίας και των λειτουργικών κοιλιακών αλγών στα παιδιά και η συσχέτισή του, με την παθογένεια, τη διάγνωση και την πρόγνωση στα παραπάνω περιοδικά σύνδρομα. Η μελέτη περιελάμβανε συνολικά 524 άτομα, 318 από αυτά ανήκαν στις 4 υπό μελέτη ομάδες παιδιών. Συγκεκριμένα η ομάδα Α περιελάμβανε 42 παιδιά με ημικρανία και κοιλιακά άλγη, η ομάδα Β 59 παιδιά με ημικρανία, η ομάδα Γ 39 παιδιά με λειτουργικά κοιλιακά άλγη και η ομάδα Δ 178 υγιή παιδιά. Τα υπόλοιπα 206 άτομα ήταν μέλη των οικογενειών των ασθενών. Μελετήθηκαν 14 πολυμορφισμοί: ΕΤ1-Κ198Ν, ΕΤ1-138InsA, ΕΤΑ-231 G/A, ETA-1363 C/T, ETB-L277L, Ser-102T/C Ser-1438A/G, MTHFR C677T, TNFα-238G/A, TNFα-308G/A, TNFα-376G/A, TNFα-857C/T, TNFα-1031C/T και ΤΝFB με τη μέθοδο PCR-RFLPs. Από τη στατιστική ανάλυση των αποτελεσμάτων υποδεικνύεται ότι: α) δεν υπάρχει κοινό γενετικό υπόστρωμα μεταξύ της ημικρανίας και των λειτουργικών κοιλιακών αλγών στα παιδιά για τους 14 πολυμορφισμούς που μελετήθηκαν, β) υπάρχει συσχέτιση του πολυμορφισμού MTHFR C677T με την ημικρανία των παιδιών της ομάδας Β, γ) υπάρχει συσχέτιση των πολυμορφισμών ΕΤΒ-L277L και ΤΝFB με τα λειτουργικά κοιλιακά άλγη των παιδιών της ομάδας Γ, δ) υπάρχει συσχέτιση του απλοτύπου [3Α]GGGGΤTA των πολυμορφισμών που εδράζονται στο χρωμόσωμα 6 (ΕΤ1-138InsA, ΕΤ1-Κ198Ν, ΤΝFα-238 G/A, TNFα-308 G/A, TNFα-376 G/A, TNFα-857 C/T , TNFα-1031 T/C, ΤΝFB) με το φαινότυπο των κοιλιακών αλγών της ομάδας Γ

Urine-Based Molecular Diagnostic Tests for Leishmaniasis Infection in Human and Canine Populations: A Meta-Analysis

Leishmaniasis is a neglected tropical disease affecting humans and domesticated animals with high mortality in endemic countries. The pleiotropy of symptoms and the complicated gold-standard methods make the need for non-invasive, highly sensitive diagnostic tests imperative. Individual studies on molecular-based Leishmania diagnosis in urine show high discrepancy; thus, a data-evidenced comparison of various techniques is necessary. We performed a systematic review and meta-analysis using the bivariate method of diagnostic methods to pool sensitivities and specificities. We investigated the impact of DNA-extraction method, PCR type, amplified locus, host species, leishmaniasis form, and geographical region. The pooled sensitivity was 69.2%. Tests performed with the kit-based DNA extraction method and qPCR outweighed in sensitivity the phenol-chloroform-based and PCR methods, while their combination showed a sensitivity of 79.3%. Amplified locus, human or canine as host and cutaneous or visceral leishmaniasis revealed similar sensitivities. Tests in European and Middle Eastern countries performed better than tests in other regions (sensitivity 81.7% vs. 43.7%). A combination of kit-based DNA extraction and qPCR could be a safer choice for molecular diagnosis for Leishmania infection in urine samples in European–Middle Eastern countries. For the rest of the world, more studies are needed to better characterize the endemic parasite species

Urine-Based Molecular Diagnostic Tests for Leishmaniasis Infection in Human and Canine Populations: A Meta-Analysis

Leishmaniasis is a neglected tropical disease affecting humans and domesticated animals with high mortality in endemic countries. The pleiotropy of symptoms and the complicated gold-standard methods make the need for non-invasive, highly sensitive diagnostic tests imperative. Individual studies on molecular-based Leishmania diagnosis in urine show high discrepancy; thus, a data-evidenced comparison of various techniques is necessary. We performed a systematic review and meta-analysis using the bivariate method of diagnostic methods to pool sensitivities and specificities. We investigated the impact of DNA-extraction method, PCR type, amplified locus, host species, leishmaniasis form, and geographical region. The pooled sensitivity was 69.2%. Tests performed with the kit-based DNA extraction method and qPCR outweighed in sensitivity the phenol-chloroform-based and PCR methods, while their combination showed a sensitivity of 79.3%. Amplified locus, human or canine as host and cutaneous or visceral leishmaniasis revealed similar sensitivities. Tests in European and Middle Eastern countries performed better than tests in other regions (sensitivity 81.7% vs. 43.7%). A combination of kit-based DNA extraction and qPCR could be a safer choice for molecular diagnosis for Leishmania infection in urine samples in European–Middle Eastern countries. For the rest of the world, more studies are needed to better characterize the endemic parasite species

Transcription factor ATF-3 regulates allele variation phenotypes of the human SLC11A1 gene

Genetic polymorphisms in the human solute carrier family 11 member 1 (SLC11A1) gene predispose to susceptibility to infectious/inflammatory diseases and cancer. Human susceptibility to these diseases exhibits allelic association with a polymorphic regulatory Z-DNA-forming microsatellite of a (GT/AC)n repeat. The carriage of different alleles may influence chromatin remodeling and accessibility by transcription factors. Of particular importance is the binding site for the Activating Protein 1 (AP-1) elements, (ATF-3 and c-Jun), adjacent to the 5’ sequence of the Z-DNA-forming polymorphism. The aim of the study was to characterize the transcriptional mechanisms controlling different alleles of SLC11A1 expression by ATF-3 and c-Jun. Allele 2, [T(GT)(5)AC(GT)(5)AC(GT)(10)GGCAGA(G)(6)], and Allele 3, [T(GT)(5)AC(GT)(5)AC(GT)(9)GGCAGA(G)(6)], were subcloned into the PGL2Basic vector. Transient transfections of THP-1 cells with the constructs, in the presence or absence of pATF-3 were preformed. Luciferase expression was determined. To document the recruitment of ATF-3 and c-Jun, to the polymorphic promoter alleles in vivo, we performed ChIP assays with transient transfected THP-1 cells treated with or without lipopolyssacharides. Our data documented that ATF-3 suppresses the transcriptional activation of Allele-3, and this suppression is enhanced in the presence of lipopolyssacharides. Our findings suggest that ATF-3 and c-Jun may influence heritable variation in SLC11A1-dependent innate resistance to infection and inflammation both within and between populations

Genetic Characterization of Carbapenem-Resistant <i>Klebsiella pneumoniae</i> Clinical Isolates in a Tertiary Hospital in Greece, 2018–2022

Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a serious public health issue. The study aimed to identify the antimicrobial resistance and accessory genes, the clonal relatedness, and the evolutionary dynamics of selected CRKP isolates recovered in an adult and pediatric intensive care unit of a tertiary hospital in Greece. Methods: Twenty-four CRKP isolates recovered during 2018–2022 were included in the study. Next-generation sequencing was performed using the Ion Torrent PGM Platform. The identification of the plasmid content, MLST, and antimicrobial resistance genes, as well as the comparison of multiple genome alignments and the identification of core genome single-nucleotide polymorphism sites, were performed using various bioinformatics software. Results: The isolates belonged to eight sequence types: 11, 15, 30, 35, 39, 307, 323, and 512. A variety of carbapenemases (KPC, VIM, NDM, and OXA-48) and resistance genes were detected. CRKP strains shared visually common genomic regions with the reference strain (NTUH-K2044). ST15, ST323, ST39, and ST11 CRKP isolates presented on average 17, 6, 16, and 866 recombined SNPs, respectively. All isolates belonging to ST15, ST323, and ST39 were classified into distinct phylogenetic branches, while ST11 isolates were assigned to a two-subclade branch. For large CRKP sets, the phylogeny seems to change approximately every seven SNPs. Conclusions: The current study provides insight into the genetic characterization of CRKP isolates in the ICUs of a tertiary hospital. Our results indicate clonal dispersion of ST15, ST323, and ST39 and highly diverged ST11 isolates

West Nile Virus in <i>Culex</i> Mosquitoes in Central Macedonia, Greece, 2022

In 2022, Greece was the second most seriously affected European country in terms of the West Nile virus (WNV), after Italy. Specifically, Central Macedonia was the region with the most reported human cases (81.5%). In the present study, 30,816 female Culex pipiens sensu lato mosquitoes were collected from May to September 2022 in the seven regional units of Central Macedonia; they were then grouped into 690 pools and tested for WNV, while next-generation sequencing was applied to the samples, which showed a cycle threshold of Ct < 30 in a real-time RT-PCR test. WNV was detected in 5.9% of pools, with significant differences in the detection rate among regional units and months. It is of interest that in the Thessaloniki regional unit, where most of the human cases were observed, the virus circulation started earlier, peaked earlier, and lasted longer than in the other regional units. All sequences clustered into the Central European subclade of WNV lineage 2, and the virus strain differed from the initial Greek strain of 2010 by 0.52% and 0.27% at the nucleotide and amino acid levels, respectively. Signature substitutions were present, such as S73P and T157A in the prM and E structural proteins, respectively. The screening of mosquitoes provides useful information for virus circulation in a region with a potential for early warning, while the availability of whole-genome sequences is essential for further studies, including virus evolution

Development of Toehold Switches as a Novel Ribodiagnostic Method for West Nile Virus

West Nile virus (WNV) is an emerging neurotropic RNA virus and a member of the genus Flavivirus. Naturally, the virus is maintained in an enzootic cycle involving mosquitoes as vectors and birds that are the principal amplifying virus hosts. In humans, the incubation period for WNV disease ranges from 3 to 14 days, with an estimated 80% of infected persons being asymptomatic, around 19% developing a mild febrile infection and less than 1% developing neuroinvasive disease. Laboratory diagnosis of WNV infection is generally accomplished by cross-reacting serological methods or highly sensitive yet expensive molecular approaches. Therefore, current diagnostic tools hinder widespread surveillance of WNV in birds and mosquitoes that serve as viral reservoirs for infecting secondary hosts, such as humans and equines. We have developed a synthetic biology-based method for sensitive and low-cost detection of WNV. This method relies on toehold riboswitches designed to detect WNV genomic RNA as transcriptional input and process it to GFP fluorescence as translational output. Our methodology offers a non-invasive tool with reduced operating cost and high diagnostic value that can be used for field surveillance of WNV in humans as well as in bird and mosquito populations