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Targeted virome enrichment and sequencing (VirCapSeq-VERT) utilizes a pool of oligos (baits) to enrich all known—up to 2015—vertebrate-infecting viruses, increasing their detection sensitivity. The hybridisation of the baits to the target sequences can be partial, thus enabling the detection and genomic reconstruction of novel pathogens with <40% genetic diversity compared to the strains used for the baits' design. In this study, we deploy this method in multiplexed mixes of viral extracts, and we assess its performance in the unbiased detection of DNA and RNA viruses after cDNA synthesis. We further assess its efficiency in depleting various background genomic material. Finally, as a proof-of-concept, we explore the potential usage of the method for the characterization of unknown, emerging human viruses, such as SARS-CoV-2, which may not be included in the baits' panel. We mixed positive samples of equimolar DNA/RNA viral extracts from SARS-CoV-2, coronavirus OC43, cytomegalovirus, influenza A virus H3N2, parvovirus B19, respiratory syncytial virus, adenovirus C and coxsackievirus A16. Targeted virome enrichment was performed on a dsDNA mix, followed by sequencing on the NextSeq500 (Illumina) and the portable MinION sequencer, to evaluate its usability as a point-of-care (PoC) application. Genome mapping assembly was performed using viral reference sequences. The untargeted libraries contained less than 1% of total reads mapped on most viral genomes, while RNA viruses remained undetected. In the targeted libraries, the percentage of viral-mapped reads were substantially increased, allowing full genome assembly in most cases. Targeted virome sequencing can enrich a broad range of viruses, potentially enabling the discovery of emerging viruses. [ABSTRACT FROM AUTHOR]

Rapid and reliable laboratory diagnosis of persons suspected of Middle East respiratory syndrome coronavirus (MERS-CoV) infection is important for timely implementation of infection control practices and disease management. In addition, monitoring molecular changes in the virus can help elucidate chains of transmission and identify mutations that might influence virus transmission efficiency. This was illustrated by a recent laboratory investigation we conducted on an imported MERS-CoV case in Greece. Two oropharyngeal swab specimens were collected on the 1st and 2nd day of patient hospitalization and tested using two real-time RT-PCR (rRT-PCR) assays targeting the UpE and Orf-1a regions of the MERS-CoV genome and RT-PCR and partial sequencing of RNA-dependent RNA polymerase and nucleocapsid genes. Serum specimens were also collected and serological test were performed. Results from the first swab sample were inconclusive while the second swab was strongly positive for MERS-CoV RNA by rRT-PCR and confirmed positive by RT-PCR and partial gene sequencing. Positive serologic test results further confirmed MERS-CoV infection. Full-length nucleocapsid and spike gene coding sequences were later obtained from the positive swab sample. Phylogenetic analysis revealed that the virus was closely related to recent human-derived MERS-CoV strains obtained in Jeddah and Makkah, Saudi Arabia, in April 2014 and dromedary camels in Saudi Arabia and Qatar. These findings were consistent with the patient’s history. We also identified a unique amino acid substitution in the spike receptor binding domain that may have implications for receptor binding efficiency. Our initial inconclusive rRT-PCR results highlight the importance of collecting multiple specimens from suspect MERS-CoV cases and particularly specimens from the lower respiratory tract. [ABSTRACT FROM AUTHOR]

The study of the human commensal micro-flora has recently regained the interest of the scientific community, particularly after the development of genomics and bio-informatics. The genes of the microbes found in the various different cavities of the human organism are collectively called the human microbiome (HM). This is a complex system, which is estimated to consist of approximately 1014 bacterial cells, thus being 10 times larger than the total of human cells. Almost 400-500 different types of microbes form the intestinal flora of the HM. The commensal bacteria in the gut microbiota interact with each other and with the host immune system, contributing to the homeostasis of the organism. The intestinal microbiome has been shown to differ among individuals of the same sex, between sexes and between individuals of different ages. Each individual human can be characterized by his/her own intestinal microbial fingerprint, which may be useful in the future for individualized medicine. Alteration in the composition and function of the commensal gut microbiota has been associated with complex diseases such as inflammatory bowel diseases (IBD), colorectal cancer, allergies and obesity. Manipulation of the gut microbiota by fecal microbiota transplantation is an effective and safe treatment of IBD and infection with C. difficile, but the use of pre- and pro-biotics for the manipulation of microbiota should be reconsidered in the light of the new information now available. This is a review of recent findings concerning the role of the enteric HM in the development of the immune system, and its connection to gut-related and non-gastroenterological diseases. [ABSTRACT FROM AUTHOR]

CagA protein contributes to pro-inflammatory responses during H. pylori infection, following its intracellular delivery to gastric epithelial cells. Here, we report for the first time in an isogenic background, on the subtle role of CagA phosphorylation on terminal EPIYA-C motifs in the transcriptional activation and expression of IL-8. We utilized isogenic H. pylori mutants of P12 reference strain, expressing CagA with varying number of EPIYA-C motifs and the corresponding phosphorylation defective EPIFA-C motifs while preserving intact the CM multimerization motifs. These mutants had been previously closely scrutinized in terms of type IV secretion system functionality, CagA translocation and its subsequent phosphorylation. Following infection of gastric epithelial cell lines, transcriptional activation of IL-8 gene and secreted IL-8 levels were found to be strictly dependent upon the functionality of the EPIYA-C phosphorylation motifs, as EPIFA-C phosphorylation-deficient CagA expression failed to induce full IL-8 transcriptional activity. Interestingly, levels of IL-8 gene activation and of secreted IL-8 were the same, irrespective of the number of EPIYA-C terminal repeats. We monitored IkBα phosphorylation and confirmed CagA involvement in NF-kB activation. Furthermore, we observed that presence of EPIYA-C functional phosphorylation motifs contributed to NF-kB activation. NF-kB upstream signaling events, such as early ERK1/2 and AKT activation were confirmed to be independent of EPIYA-C phosphorylation. On the contrary, use of TAK1 specific inhibitor 5Z-7-Oxozeaenol resulted in complete arrest of IL-8 secretion, in a dose-dependent manner, irrespective of CagA status. H. pylori-infected TAK1-/- mouse embryonic fibroblasts (MEFs) failed to induce NF-kB activity, unlike the respective control MEFs. CagA and TAK1 were found to immunoprecipitate together, irrespective of CagA EPIYA-C status, thus confirming earlier reports of TAK1 and CagA protein interaction. Our data suggest that CagA may potentially interfere with TAK1 activity during NF-kB activation for IL-8 induction in early H. pylori infection. [ABSTRACT FROM AUTHOR]

In the literature, miracles are often placed in a position antagonistic to conventional medicine, and are co-examined in the context of "folklore medical wisdom". Despite the fact that in later years the study of the lives of saints and of their miracles has attracted the interest of historians, it is debatable whether their miracles could be considered as a specific therapeutic practice, entirely discrete from traditional "folklore" medical wisdom. This article aims to show that the medical practice as exercised by the Church fully recognizes the classical medical science of that era, which in this light can be seen to offer what has been described as "holistic medicine". Elements of different categories of miracles are reported, with a special emphasis on miracles in specific groups, such as women, children and elderly people, along with examples from various different nosological entities, such as psychiatric, neurological and ophthalmological miracles and transplantations. The reports of miracles, in combination with the progress of medicine in Byzantium provide evidence that lay people in the Byzantine epoch had a very deep concern for the body and its cure from pathological conditions and diseases. [ABSTRACT FROM AUTHOR]

A clinical isolate of Klebsiella pneumoniae resistant to a wide variety of beta-lactams, including third generation cephalosporins, aztreonam and cephamycins, as well as to beta-lactam/clavulanate and sulbactam combinations was examined. It was found that this resistance was transmissible to Escherichia coli recipients via a small 5.3 MDa plasmid encoding for an unusual beta-lactamase produced in relatively large quantities. The enzyme, designated LAT-1, exhibited a highly basic isoelectric point (pI = 9.4) and its hydrolytic activity resembled closely that of a class-I chromosomal cephalosporinase. In vitro, LAT-1 hydrolysed cephaloridine, cephalothin and cephalexin more rapidly than penicillins. A slow hydrolysis of cefoxitin, ceftibuten, ceftazidime and cefotaxime was also observed. The enzyme was inhibited by low concentrations of aztreonam and cloxacillin but it was virtually unaffected by clavulanate. Under stringent conditions, the LAT-1 encoding plasmid did not hybridize with probes specific for TEM-1 and Enterobacter cloacae AmpC beta-lactamase genes. The plasmid was not self-transferable but was readily mobilized by a conjugative R-plasmid harboured by the same K. pneumoniae strain. [ABSTRACT FROM AUTHOR]

A clinical isolate of resistant to a wide variety of -lactams, including third generation cephalosporins, aztreonam and cephamycins, as well as to -lactam/clavulanate and sulbactam combinations was examined. It was found that this resistance was transmissible to recipients via a small 5·3 MDa plasmid encoding for an unusual -lactamase produced in relatively large quantities. The enzyme, designated LAT-1, exhibited a highly basic isoelectric point (pI = 9·4) and its hydrolytic activity resembled closely that of a class-I chromosomal cephalosporinase. , LAT-1 hydrolysed cephaloridine, cephalothin and cephalexin more rapidly than penicillins. A slow hydrolysis of cefoxitin, ceftibuten, ceftazidime and cefotaxime was also observed. The enzyme was inhibited by low concentrations of aztreonam and cloxacillin but it was virtually unaffected by clavulanate. Under stringent conditions, the LAT-1 encoding plasmid did not hybridize with probes specific for TEM-1 and AmpC -lactamase genes. The plasmid was not self-transferable but was readily mobilized by a conjugative R-plasmid harboured by the same strain. [ABSTRACT FROM PUBLISHER]

Background Head injuries (HI) as a result of falls in winter sport activities, despite their incidence is not very high, they are due to their high severity of lesion, frequently associated with severe disabilities and complicated rehabilitations. Methods The magnitude of head injuries due to falls among alpine skiers and snowboarders (HIFASS) in Slovenia over the last decade were assessed using multiple nationwide Slovenian data. Hospitalisation-, emergency visitation- and mortality were calculated and compared to data of ski slope injuries. Differences by gender, age and injury were assessed, and annual changes were calculated by poisson regression analysis. Results 78.083 hospitalisations, 98.557 emergency visits and 2.969 deaths due to HI were registered in the observed period, and the HIFASS were minimally represented among them (1.7% hospitalisations, 0.6% emergency visits and 0.3% deaths). Although the decreasing trend for all HI (annual change −2.1%, p=0.0001), HIFASS had an increasing secular trend (annual change +3%, p=0.001). Individuals younger than 40 years were the most affected and had also mostly severe intracranial lesions or bone fractures. The incidence of HIFASS was about 0.2 per 1000 skier-days. Conclusion Severe, even fatal HIFASS, may be prevented by carrying a helmet. Slovenia has mandatory helmet use legislation for children until 14 years of age. But take into account results of evidence-based systematic analyses, the obligatory helmet use should include all age groups. [ABSTRACT FROM PUBLISHER]

Hand amputations constitute a major cause of disability and public health concern with significant socio-economic dimensions. Aim To investigate hand amputation epidemiology, identify risk groups and estimate the potential for prevention. Methods EDISS in Greece, comprising four hospitals across the country, captures patient and injury information (demographics, accident conditions, injury description, outcome). During 1996–2004, among ∼0.5 million cases recorded, 86 211 were hand injuries resulting in 964 amputations (1.1%). To identify preventable amputation causes detailed injury descriptions regarding injury mechanism were examined. Results Among hand amputations 937 involved fingers, 23 palms and 4 wrists. The estimated (extrapolated) number of amputations was 3940 and the calculated overall incidence rate (IR) 3.62 per 10 000 person-years. The highest IR (8.02) was recorded among males 15–64 years old. The three most common mechanisms involved: tools in 445 (57.8%), doors in 172 (22.3%) and materials in 50 (6.5%) cases; of note, 148 victims were woodworkers (15.3%). The least common causes were animals, jewellery and fireworks (10, 7 and 6 cases respectively). Analysis of the accident conditions revealed the most common causes: lack of preventive measures/appropriate training and work overload. Most amputations due to doors were attributed to non-use of door handle while closing the door or could have been prevented by a chocking door closing mechanism. Conclusions Amputations of the hand constitute a small but non-negligible percentage of all hand injuries. The main risk group is young males and woodwork professionals, whereas doors pose a significant risk. Raising awareness and implementing simple measures could reduce the burden of this disabilitating injury. [ABSTRACT FROM PUBLISHER]

In the context of poliomyelitis eradication, a reinforced supplementary laboratory surveillance of enteroviruses was implemented in Greece. Between 2008 and 2014, the Hellenic Polioviruses/Enteroviruses Reference Laboratory performed detailed supplementary surveillance of circulating enteroviruses among healthy individuals in high-risk population groups, among immigrants from countries in which poliovirus is endemic, and in environmental samples. In total, 722 stool samples and 179 sewage water samples were included in the study. No wild-type polioviruses were isolated during these 7 years of surveillance, although two imported vaccine polioviruses were detected. Enterovirus presence was recorded in 25.3 and 25.1% of stool and sewage water samples, respectively. Nonpolio enteroviruses isolated from stool samples belonged to species A, B, or C; coxsackievirus A24 was the most frequently identified serotype. Only enteroviruses of species B were identified in sewage water samples, including four serotypes of echoviruses and four serotypes of coxsackie B viruses. Phylogenetic analysis revealed close genetic relationships among virus isolates from sewage water samples and stool samples, which in most cases fell into the same cluster. To the best of our knowledge, this is the first study to compare enterovirus serotypes circulating in fecal specimens of healthy individuals and environmental samples, emphasizing the burden of enterovirus circulation in asymptomatic individuals at high risk. Given that Greece continues to receive a large number of short-term arrivals, students, migrants, and refugees from countries in which poliovirus is endemic, it is important to guarantee high-quality surveillance in order to maintain its polio-free status until global eradication is achieved. [ABSTRACT FROM AUTHOR]

Low oxygen tension exerts a significant effect on the replication of several DNA and RNA viruses in cultured cells. In vitro propagation of hepatitis C virus (HCV) has thus far been studied under atmospheric oxygen levels despite the fact that the liver tissue microenvironment is hypoxic. In this study, we investigated the efficiency of HCV production in actively dividing or differentiating human hepatoma cells cultured under low or atmospheric oxygen tensions. By using both HCV replicons and infection-based assays, low oxygen was found to enhance HCV RNA replication whereas virus entry and RNA translation were not affected. Hypoxia signaling pathway-focused DNA microarray and real-time quantitative reverse transcription-PCR (qRT-PCR) analyses revealed an upregulation of genes related to hypoxic stress, glycolytic metabolism, cell growth, and proliferation when cells were kept under low (3% [vol/vol]) oxygen tension, likely reflecting cell adaptation to anaerobic conditions. Interestingly, hypoxia-mediated enhancement of HCV replication correlated directly with the increase in anaerobic glycolysis and creatine kinase B (CKB) activity that leads to elevated ATP production. Surprisingly, activation of hypoxia-inducible factor alpha (HIF-α) was not involved in the elevation of HCV replication. Instead, a number of oncogenes known to be associated with glycolysis were upregulated and evidence that these oncogenes contribute to hypoxia-mediated enhancement of HCV replication was obtained. Finally, in liver biopsy specimens of HCV-infected patients, the levels of hypoxia and anaerobic metabolism markers correlated with HCV RNA levels. These results provide new insights into the impact of oxygen tension on the intricate HCV-host cell interaction. [ABSTRACT FROM AUTHOR]

Abstract: Streptococcus macedonicus ACA-DC 198, a bacteriocin producer isolated from Greek Kasseri cheese, was used in a series of in vitro and in vivo experiments in order to evaluate its pathogenic potential. The strain was examined in vitro for haemolytic activity, antibiotic resistance and presence of pathogenicity genes encountered in Streptococcus pyogenes. Subsequently, the strain was orally administered to mice (8.9 log cfu daily), continuously over a period of 12 weeks, in order to ascertain the effects of its long term consumption on animal health and gastric inflammation. S. macedonicus ACA-DC 198 was found to be non-haemolytic and sensitive to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, streptomycin, tetracycline, and vancomycin, with the only resistance observed against kanamycin. PCR amplification and DNA–DNA hybridization did not reveal the presence of any of the S. pyogenes pathogenicity genes examined, namely emm, scpA, hasA, speB, smez2, speJ, sagAB, hylA, ska, speF, speG, slo, hylP2 and mga. In the mouse study, no detrimental effects were observed in the behaviour, general well being, weight gain and water consumption of the animals receiving S. macedonicus ACA-DC 198. Histologic analysis showed no evidence of inflammation in the stomach of the animals receiving S. macedonicus ACA-DC 198, while faecal microbiological analysis revealed that the strain retained its viability passing through the mouse gastrointestinal tract. Finally, no evidence of translocation to the liver, spleen and mesenteric lymph nodes was observed. In conclusion, none of the examined virulence determinants were detected in S. macedonicus ACA-DC 198 and its long term, high dosage oral administration did not appear to induce any pathogenic effect in mice. [Copyright &y& Elsevier]

• EV surveillance studies are scarce in Greece. • We provide a comprehensive view of EV circulation in hospitalized patients in Greece during 2013−15. • Echo30 was the most frequent EV-B serotype whereas CoxA6 the most frequent EV-A one. • Most EV strains from Greece were similar to strains circulating in Europe during the same period. There are only sporadic data for the circulation of Enteroviruses (EVs) in Greece with previous studies reporting mainly the presence of Echoviruses (E) and Coxsackie viruses (CV) B. We carried out a surveillance study for the molecular characterization of EVs detected in hospitalized patients throughout Greece as well as a phylogenetic analysis of the most frequently encountered serotypes. Stools, cerebrospinal fluids, throat swabs and blood samples were collected from hospitalized patients with suspicion of EV infection. All samples were tested for EVs by rRT-PCR targeting the 5′ untranslated region of EV genome. For positive samples, PCR amplification and sequencing targeting a part of VP1 region was performed. We examined 831 samples and 209 were positive for EVs with Enterovirus B species being the most frequently amplified. E30, CVB5 and E9 were the most frequent serotypes of Enterovirus B species, whereas CVA6 and EV-A71 the most frequent serotypes of Enterovirus A species. Evs were significantly detected more frequently in stool samples compared to other types of specimens. Phylogenetic analysis revealed that most EV-A71 strains clustered in the subgenogroups C2 whereas all the CVA6 strains belonged to sub-genotype D3. Additionally, two different lineages of E30 and three different clusters of E9 viruses circulated simultaneously in Greece. Our data indicated that most EV strains from Greece were similar to strains circulating throughout Europe during the same period. We provide a comprehensive picture of EVs circulating in Greece which can be helpful to interpret trends in EV diseases by associating them with circulating serotypes. [ABSTRACT FROM AUTHOR]