AAV9-mediated SH3TC2 gene replacement therapy targeted to Schwann cells for the treatment of CMT4C.

Type 4C Charcot-Marie-Tooth (CMT4C) demyelinating neuropathy is caused by autosomal recessive SH3TC2 gene mutations. SH3TC2 is highly expressed in myelinating Schwann cells. CMT4C is a childhood-onset progressive disease without effective treatment. Here, we generated a gene therapy for CMT4C mediated by an adeno-associated viral 9 vector (AAV9) to deliver the human SH3TC2 gene in the Sh3tc2-/- mouse model of CMT4C. We used a minimal fragment of the myelin protein zero (Mpz) promoter (miniMpz), which was cloned and validated to achieve Schwann cell-targeted expression of SH3TC2. Following the demonstration of AAV9-miniMpz.SH3TC2myc vector efficacy to re-establish SH3TC2 expression in the peripheral nervous system, we performed an early as well as a delayed treatment trial in Sh3tc2-/- mice. We demonstrate both after early as well as following late treatment improvements in multiple motor performance tests and nerve conduction velocities. Moreover, treatment led to normalization of the organization of the nodes of Ranvier, which is typically deficient in CMT4C patients and Sh3tc2-/- mice, along with reduced ratios of demyelinated fibers, increased myelin thickness and reduced g-ratios at both time points of intervention. Taken together, our results provide a proof of concept for an effective and potentially translatable gene replacement therapy for CMT4C treatment.

Cryptomphalus aspersa Egg Extract Protects against Human Stem Cell Stress-Induced Premature Senescence.

Cellular senescence is a tightly regulated pathophysiologic process and is caused by replicative exhaustion or external stressors. Since naturally derived bioactive compounds with anti-ageing properties have recently captured scientific interest, we analysed the anti-ageing and antioxidant efficacy of Cryptomphalus aspersa egg extract (CAEE). Its effects on stemness, wound-healing properties, antioxidant defense mechanisms, and DNA damage repair ability of Human Wharton's jelly mesenchymal stem cells (WJ-MSCs) were analysed. Our results revealed that CAEE fortifies WJ-MSCs stemness, which possibly ameliorates their wound-healing ability. Additionally, we show that CAEE possesses a strong antioxidant capacity as demonstrated by the elevation of the levels of the basic antioxidant molecule, GSH, and the induction of the NRF2, a major antioxidant regulator. In addition, CAEE alleviated cells' oxidative stress and therefore prevented stress-induced premature senescence (SIPS). Furthermore, we demonstrated that the prevention of SIPS could be mediated via the extract's ability to induce autophagy, as indicated by the elevation of the protein levels of all basic autophagic molecules and the increase in formation of autophagolysosomes in CAEE-treated WJ-MSCs. Moreover, CAEE-treated cells exhibited decreased Caveolin-1 levels. We propose that Cryptomphalus aspersa egg extract comprises bioactive compounds that can demonstrate strong antioxidant/anti-ageing effects by regulating the Caveolin-1-autophagy-senescence molecular axis.

Multi-omics data integration and network-based analysis drives a multiplex drug repurposing approach to a shortlist of candidate drugs against COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is undeniably the most severe global health emergency since the 1918 Influenza outbreak. Depending on its evolutionary trajectory, the virus is expected to establish itself as an endemic infectious respiratory disease exhibiting seasonal flare-ups. Therefore, despite the unprecedented rally to reach a vaccine that can offer widespread immunization, it is equally important to reach effective prevention and treatment regimens for coronavirus disease 2019 (COVID-19). Contributing to this effort, we have curated and analyzed multi-source and multi-omics publicly available data from patients, cell lines and databases in order to fuel a multiplex computational drug repurposing approach. We devised a network-based integration of multi-omic data to prioritize the most important genes related to COVID-19 and subsequently re-rank the identified candidate drugs. Our approach resulted in a highly informed integrated drug shortlist by combining structural diversity filtering along with experts' curation and drug-target mapping on the depicted molecular pathways. In addition to the recently proposed drugs that are already generating promising results such as dexamethasone and remdesivir, our list includes inhibitors of Src tyrosine kinase (bosutinib, dasatinib, cytarabine and saracatinib), which appear to be involved in multiple COVID-19 pathophysiological mechanisms. In addition, we highlight specific immunomodulators and anti-inflammatory drugs like dactolisib and methotrexate and inhibitors of histone deacetylase like hydroquinone and vorinostat with potential beneficial effects in their mechanisms of action. Overall, this multiplex drug repurposing approach, developed and utilized herein specifically for SARS-CoV-2, can offer a rapid mapping and drug prioritization against any pathogen-related disease.

Serum Reactive Antibodies against the N-Methyl-D-Aspartate Receptor NR2 Subunit-Could They Act as Potential Biomarkers?

Synaptic dysfunction and disrupted communication between neuronal and glial cells play an essential role in the underlying mechanisms of multiple sclerosis (MS). Earlier studies have revealed the importance of glutamate receptors, particularly the N-methyl-D-aspartate (NMDA) receptor, in excitotoxicity, leading to abnormal synaptic transmission and damage of neurons. Our study aimed to determine whether antibodies to the NR2 subunit of NMDAR are detected in MS patients and evaluate the correlation between antibody presence and clinical outcome. Furthermore, our focus extended to examine a possible link between NR2 reactivity and anti-coagulant antibody levels as pro-inflammatory molecules associated with MS. A cross-sectional study was carried out, including 95 patients with MS and 61 age- and gender-matched healthy controls (HCs). The enzyme-linked immunosorbent assay was used to detect anti-NR2 antibodies in serum samples of participants along with IgG antibodies against factor (F)VIIa, thrombin, prothrombin, FXa, and plasmin. According to our results, significantly elevated levels of anti-NR2 antibodies were detected in MS patients compared to HCs (p < 0.05), and this holds true when we compared the Relapsing-Remitting MS course with HCs (p < 0.05). A monotonically increasing correlation was found between NR2 seropositivity and advanced disability (rs = 0.30; p < 0.01), anti-NR2 antibodies and disease worsening (rs = 0.24; p < 0.05), as well as between antibody activity against NR2 and thrombin (rs = 0.33; p < 0.01). The presence of anti-NR2 antibodies in MS patients was less associated with anti-plasmin IgG antibodies [OR:0.96 (95%CI: 0.92-0.99); p < 0.05]; however, such an association was not demonstrated when analyzing only RRMS patients. In view of our findings, NR2-reactive antibodies may play, paving the way for further research into their potential as biomarkers and therapeutic targets in MS.

A Comparison of Two Analytical Approaches for the Quantification of Neurofilament Light Chain, a Biomarker of Axonal Damage in Multiple Sclerosis.

Neurofilament light chain (NfL), is a neuron-specific cytoskeletal protein detected in extracellular fluid following axonal damage. Extensive research has focused on NfL quantification in CSF, establishing it as a prognostic biomarker of disability progression in Multiple Sclerosis (MS). Our study used a new commercially available Enzyme-Linked Immunosorbent Assay (ELISA) kit and Single Molecular Array (Simoa) advanced technology to assess serum NfL levels in MS patients and Healthy Controls (HC). Verifying the most accurate, cost-effective methodology will benefit its application in clinical settings. Blood samples were collected from 54 MS patients and 30 HC. Protocols accompanying the kits were followed. The ELISA thershold was set as 3 S.D. above the mean of the HC. For Simoa, the Z-score calculation created by Jens Kuhle's group was applied (with permission). Samples exceeding the threshold or z-score ≥1.5 indicated subclinical disease activity. To our knowledge, this is the first study to find strong-positive correlation between ELISA and Simoa for the quantification of NfL in serum (r = 0.919). Despite the strong correlation, Simoa has better analytical sensitivity and can detect small changes in samples making it valuable in clinical settings. Further research is required to evaluate whether serum NfL quantification using ELISA could be utilized to predict disability progression.

AAV9-mediated Schwann cell-targeted gene therapy rescues a model of demyelinating neuropathy.

Mutations in the GJB1 gene, encoding the gap junction (GJ) protein connexin32 (Cx32), cause X-linked Charcot-Marie-Tooth disease (CMT1X), an inherited demyelinating neuropathy. We developed a gene therapy approach for CMT1X using an AAV9 vector to deliver the GJB1/Cx32 gene under the myelin protein zero (Mpz) promoter for targeted expression in Schwann cells. Lumbar intrathecal injection of the AAV9-Mpz.GJB1 resulted in widespread biodistribution in the peripheral nervous system including lumbar roots, sciatic and femoral nerves, as well as in Cx32 expression in the paranodal non-compact myelin areas of myelinated fibers. A pre-, as well as post-onset treatment trial in Gjb1-null mice, demonstrated improved motor performance and sciatic nerve conduction velocities along with improved myelination and reduced inflammation in peripheral nerve tissues. Blood biomarker levels were also significantly ameliorated in treated mice. This study provides evidence that a clinically translatable AAV9-mediated gene therapy approach targeting Schwann cells could potentially treat CMT1X.

Gene replacement therapy in a model of Charcot-Marie-Tooth 4C neuropathy.

Charcot-Marie-Tooth disease type 4C is the most common recessively inherited demyelinating neuropathy that results from loss of function mutations in the SH3TC2 gene. Sh3tc2-/- mice represent a well characterized disease model developing early onset progressive peripheral neuropathy with hypo- and demyelination, slowing of nerve conduction velocities and disturbed nodal architecture. The aim of this project was to develop a gene replacement therapy for treating Charcot-Marie-Tooth disease type 4C to rescue the phenotype of the Sh3tc2-/- mouse model. We generated a lentiviral vector LV-Mpz.SH3TC2.myc to drive expression of the human SH3TC2 cDNA under the control of the Mpz promoter specifically in myelinating Schwann cells. The vector was delivered into 3-week-old Sh3tc2-/- mice by lumbar intrathecal injection and gene expression was assessed 4-8 weeks after injection. Immunofluorescence analysis showed presence of myc-tagged human SH3TC2 in sciatic nerves and lumbar roots in the perinuclear cytoplasm of a subset of Schwann cells, in a dotted pattern co-localizing with physiologically interacting protein Rab11. Quantitative PCR analysis confirmed SH3TC2 mRNA expression in different peripheral nervous system tissues. A treatment trial was initiated in 3 weeks old randomized Sh3tc2-/- littermate mice which received either the full or mock (LV-Mpz.Egfp) vector. Behavioural analysis 8 weeks after injection showed improved motor performance in rotarod and foot grip tests in treated Sh3tc2-/- mice compared to mock vector-treated animals. Moreover, motor nerve conduction velocities were increased in treated Sh3tc2-/- mice. On a structural level, morphological analysis revealed significant improvement in g-ratios, myelin thickness, and ratios of demyelinated fibres in lumbar roots and sciatic nerves of treated Sh3tc2-/- mice. Finally, treated mice also showed improved nodal molecular architecture and reduction of blood neurofilament light levels, a clinically relevant biomarker for axonal injury/degeneration. This study provides a proof of principle for viral gene replacement therapy targeted to Schwann cells to treat Charcot-Marie-Tooth disease type 4C and potentially other similar demyelinating inherited neuropathies.

Intrathecal gene therapy rescues a model of demyelinating peripheral neuropathy.

Inherited demyelinating peripheral neuropathies are progressive incurable diseases without effective treatment. To develop a gene therapy approach targeting myelinating Schwann cells that can be translatable, we delivered a lentiviral vector using a single lumbar intrathecal injection and a myelin-specific promoter. The human gene of interest, GJB1, which is mutated in X-linked Charcot-Marie-Tooth Disease (CMT1X), was delivered intrathecally into adult Gjb1-null mice, a genetically authentic model of CMT1X that develops a demyelinating peripheral neuropathy. We obtained widespread, stable, and cell-specific expression of connexin32 in up to 50% of Schwann cells in multiple lumbar spinal roots and peripheral nerves. Behavioral and electrophysiological analysis revealed significantly improved motor performance, quadriceps muscle contractility, and sciatic nerve conduction velocities. Furthermore, treated mice exhibited reduced numbers of demyelinated and remyelinated fibers and fewer inflammatory cells in lumbar motor roots, as well as in the femoral motor and sciatic nerves. This study demonstrates that a single intrathecal lentiviral gene delivery can lead to Schwann cell-specific expression in spinal roots extending to multiple peripheral nerves. This clinically relevant approach improves the phenotype of an inherited neuropathy mouse model and provides proof of principle for treating inherited demyelinating neuropathies.

Gene therapy targeting oligodendrocytes provides therapeutic benefit in a leukodystrophy model.

Pelizaeus-Merzbacher-like disease or hypomyelinating leukodystrophy-2 is an autosomal recessively inherited leukodystrophy with childhood onset resulting from mutations in the gene encoding the gap junction protein connexin 47 (Cx47, encoded by GJC2). Cx47 is expressed specifically in oligodendrocytes and is crucial for gap junctional communication throughout the central nervous system. Previous studies confirmed that a cell autonomous loss-of-function mechanism underlies hypomyelinating leukodystrophy-2 and that transgenic oligodendrocyte-specific expression of another connexin, Cx32 (GJB1), can restore gap junctions in oligodendrocytes to achieve correction of the pathology in a disease model. To develop an oligodendrocyte-targeted gene therapy, we cloned the GJC2/Cx47 gene under the myelin basic protein promoter and used an adeno-associated viral vector (AAV.MBP.Cx47myc) to deliver the gene to postnatal Day 10 mice via a single intracerebral injection in the internal capsule area. Lasting Cx47 expression specifically in oligodendrocytes was detected in Cx47 single knockout and Cx32/Cx47 double knockout mice up to 12 weeks post-injection, including the corpus callosum and the internal capsule but also in more distant areas of the cerebrum and in the spinal cord. Application of this oligodendrocyte-targeted somatic gene therapy at postnatal Day 10 in groups of double knockout mice, a well characterized model of hypomyelinating leukodystrophy-2, resulted in significant improvement in motor performance and coordination at 1 month of age in treated compared to mock-treated mice, as well as prolonged survival. Furthermore, immunofluorescence and morphological analysis revealed improvement in demyelination, oligodendrocyte apoptosis, inflammation, and astrogliosis, all typical features of this leukodystrophy model in both brain and spinal cord. Functional dye transfer analysis confirmed the re-establishment of oligodendrocyte gap junctional connectivity in treated as opposed to untreated mice. These results provide a significant advance in the development of oligodendrocyte-cell specific gene therapy. Adeno-associated viral vectors can be used to target therapeutic expression of a myelin gene to oligodendrocytes. We show evidence for the first somatic gene therapy approach to treat hypomyelinating leukodystrophy-2 preclinically, providing a potential treatment for this and similar forms of leukodystrophies.

HPV prevalence and type distribution in Cypriot women with cervical cytological abnormalities.

BACKGROUND: Human papillomavirus (HPV) is the most common sexually transmitted agent, and it can cause cervical lesions and cancer in females. Currently, information regarding the prevalence of HPV in Cyprus is lacking. The aim of this study was to evaluate the HPV type-specific prevalence in 596 women, aged 19-65 years, with cytological abnormalities. Additionally, in a subset of 348 women for whom cytology results of the Pap test were available, the association between HPV infection and cervical disease was investigated. METHODS: HPV detection and typing was carried out using PCR and restriction fragment length polymorphism analysis, respectively. RESULTS: Overall, the HPV prevalence was 72.8%, and it was shown to be age dependent, with a decreasing prevalence until the age of 45 years (p = 0.0018, χ2). Two hundred and fifty-eight women (59.4%) were infected with high-risk HPV, 151 (34.8%) with low-risk HPV, and 25 (5.8%) with HPV types of unknown risk. The most common high-risk HPV type was HPV16 (17.7%), followed by HPV31 (12.9%), HPV58 (7.1%), HPV68 (4.6%), HPV18 (4.1%), and HPV56 (3.7%). Among the women for whom cytology results were available, 268 (77%) were HPV positive, with a sample distribution as follows: 188 (74%) had atypical squamous cells of undetermined significance (ASCUS), 61 (85.9%) had low-grade squamous intraepithelial lesion (L-SIL), and 19 (82.6%) had high-grade squamous intraepithelial lesion (H-SIL). HPV16 was the most common type among women affected by L-SIL (19.7%) and H-SIL (15.8%), with HPV31 being the most common type in women affected by ASCUS (16.5%). CONCLUSIONS: The present study provides the first epidemiological data related to HPV prevalence and type distribution in Cypriot women with cytological abnormalities.

Intraneural GJB1 gene delivery improves nerve pathology in a model of X-linked Charcot-Marie-Tooth disease.

OBJECTIVE: X-linked Charcot-Marie-Tooth disease (CMT1X) is a common inherited neuropathy caused by mutations in the GJB1 gene encoding the gap junction protein connexin32 (Cx32). Clinical studies and disease models indicate that neuropathy mainly results from Schwann cell autonomous, loss-of-function mechanisms; therefore, CMT1X may be treatable by gene replacement. METHODS: A lentiviral vector LV.Mpz-GJB1 carrying the GJB1 gene under the Schwann cell-specific myelin protein zero (Mpz) promoter was generated and delivered into the mouse sciatic nerve by a single injection immediately distal to the sciatic notch. Enhanced green fluorescent protein (EGFP) reporter gene expression was quantified and Cx32 expression was examined on a Cx32 knockout (KO) background. A gene therapy trial was performed in a Cx32 KO model of CMT1X. RESULTS: EGFP was expressed throughout the length of the sciatic nerve in up to 50% of Schwann cells starting 2 weeks after injection and remaining stable for up to 16 weeks. Following LV.Mpz-GJB1 injection into Cx32 KO nerves, we detected Cx32 expression and correct localization in non-compact myelin areas where gap junctions are normally formed. Gene therapy trial by intraneural injection in groups of 2-month-old Cx32 KO mice, before demyelination onset, significantly reduced the ratio of abnormally myelinated fibers (p = 0.00148) and secondary inflammation (p = 0.0178) at 6 months of age compared to mock-treated animals. INTERPRETATION: Gene delivery using a lentiviral vector leads to efficient gene expression specifically in Schwann cells. Restoration of Cx32 expression ameliorates nerve pathology in a disease model and provides a promising approach for future treatments of CMT1X and other inherited neuropathies.

Gene delivery targeted to oligodendrocytes using a lentiviral vector.

BACKGROUND: Most leukodystrophies result from mutations in genes expressed in oligodendrocytes that may cause autonomous loss of function of cell structural proteins. Therefore, effective gene delivery to oligodendrocytes is necessary to develop future treatments. MATERIALS: To achieve this, we cloned a lentiviral vector in which the enhanced green fluorescent protein (EGFP) expression was driven by the oligodendrocyte specific 2,3-cyclic nucleotide 3-phosphodiesterase promoter. The vector was inserted into C57BL/6 neonatal mouse brain by combined intraventricular and parenchymal injections. RESULTS: Assessment of EGFP expression revealed a widespread distribution, specifically in cells of the oligodendrocyte linage, starting from postnatal day 6 (P6) in the subventricular zone and spreading through migrating oligodendrocyte precursors. By P30, it was detectable throughout the brain and persisted for at least 3 months, showing an increase both in the number of expressing cells and in intensity over time. EGFP expression was restricted to oligodendrocyte linage cells. On average, 20.3 ± 2.56% of all oligodendrocytes in different central nervous system areas were EGFP-positive, with regional variations. CONCLUSIONS: Lentiviral gene delivery using an oligodendrocyte-specific promoter may achieve widespread and long-lasting expression selectively in oligodendrocytes, offering a possibility for gene therapy in certain leukodystrophies, although the relatively low rates of oligodendrocyte transduction are a limitation that remains to be overcome.

Phylogenetic patterns of human coxsackievirus B5 arise from population dynamics between two genogroups and reveal evolutionary factors of molecular adaptation and transmission.

The aim of this study was to gain insights into the tempo and mode of the evolutionary processes that sustain genetic diversity in coxsackievirus B5 (CVB5) and into the interplay with virus transmission. We estimated phylodynamic patterns with a large sample of virus strains collected in Europe by Bayesian statistical methods, reconstructed the ancestral states of genealogical nodes, and tested for selection. The genealogies estimated with the structural one-dimensional gene encoding the VP1 protein and nonstructural 3CD locus allowed the precise description of lineages over time and cocirculating virus populations within the two CVB5 clades, genogroups A and B. Strong negative selection shaped the evolution of both loci, but compelling phylogenetic data suggested that immune selection pressure resulted in the emergence of the two genogroups with opposed evolutionary pathways. The genogroups also differed in the temporal occurrence of the amino acid changes. The virus strains of genogroup A were characterized by sequential acquisition of nonsynonymous changes in residues exposed at the virus 5-fold axis. The genogroup B viruses were marked by selection of three changes in a different domain (VP1 C terminus) during its early emergence. These external changes resulted in a selective sweep, which was followed by an evolutionary stasis that is still ongoing after 50 years. The inferred population history of CVB5 showed an alternation of the prevailing genogroup during meningitis epidemics across Europe and is interpreted to be a consequence of partial cross-immunity.

Acute hepatitis and myositis associated with Erythema infectiosum by Parvovirus B19 in an adolescent.

BACKGROUND: Erythema infectiosum is the most common clinical manifestation of Parvovirus B19 infection although it has also been associated with rheumatologic diseases and various types of systemic vasculitides. Acute hepatitis and benign myositis however are rarely reported in association with Parvovirus B19 infection. CASE PRESENTATION: Here we report a 14-year old male, who developed acute hepatitis and benign myositis associated with erythema infectiosum following Parvovirus B19 infection. CONCLUSION: Parvovirus B19 infection has rarely been associated with acute hepatitis and exceptionally rarely with benign myositis. Parvovirus B19 should be considered in the differential diagnosis of acute non-A to E hepatitis and in the case of acute benign myositis presenting with a rash especially in children.

Orally Administered Drugs and Their Complicated Relationship with Our Gastrointestinal Tract.

Orally administered compounds represent the great majority of all pharmaceutical compounds produced for human use and are the most popular among patients since they are practical and easy to self-administer. Following ingestion, orally administered drugs begin a "perilous" journey down the gastrointestinal tract and their bioavailability is modulated by numerous factors. The gastrointestinal (GI) tract anatomy can modulate drug bioavailability and accounts for interpatient drug response heterogeneity. Furthermore, host genetics is a contributor to drug bioavailability modulation. Importantly, a component of the GI tract that has been gaining notoriety with regard to drug treatment interactions is the gut microbiota, which shares a two-way interaction with pharmaceutical compounds in that they can be influenced by and are able to influence administered drugs. Overall, orally administered drugs are a patient-friendly treatment option. However, during their journey down the GI tract, there are numerous host factors that can modulate drug bioavailability in a patient-specific manner.

SARS-CoV-2-specific antibody responses following BNT162b2 vaccination in individuals with multiple sclerosis receiving different disease-modifying treatments.

Introduction: The study aims to evaluate the concentration of IgG antibodies against the receptor-binding domain of the SARS-CoV-2 spike1 protein (S1RBD) in BNT162b2- vaccinated relapsing-remitting multiple sclerosis (RRMS) individuals receiving disease-modifying treatments (DMTs). Methods: Serum from 126 RRMS volunteers was collected 3 months after the administration of the second dose of the Pfizer-BioNTech BNT162b2 vaccine. Additional samples were analyzed after the administration of the booster dose in fingolimod- treated MS. Anti-S1RBD IgG antibody concentrations were quantified using the ABBOTT SARS-CoV-2 IgG II Quant assay. Results: Anti-S1RBD IgG antibody concentrations in RRMS individuals receiving natalizumab, interferons, teriflunomide, and dimethyl fumarate showed no significant difference to those in healthy controls. However, fingolimod-treated MS individuals showed a marked inability to produce SARS-CoV-2- specific antibodies (p < 0.0001). Furthermore, a booster dose was not able to elicit the production of IgG antibodies in a large portion of matched individuals. Discussion: A possible explanation for the altered immune response in fingolimod- treated MS individuals could be due to the medication inhibiting the circulation of lymphocytes, and possibly in turn inhibiting antibody production. Overall, patients on DMTs are generally of no disadvantage toward mounting an immune response against the vaccine. Nevertheless, further studies require evaluating non-humoral immunity against SARS-CoV-2 following vaccination, as well as the suitability of such vaccinations on patients treated with fingolimod.

Detection of Circulating SARS-CoV-2 Variants of Concern (VOCs) Using a Multiallelic Spectral Genotyping Assay.

Throughout the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continuously evolved, resulting in new variants, some of which possess increased infectivity, immune evasion, and virulence. Such variants have been denoted by the World Health Organization as variants of concern (VOC) because they have resulted in an increased number of cases, posing a strong risk to public health. Thus far, five VOCs have been designated, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529), including their sublineages. Next-generation sequencing (NGS) can produce a significant amount of information facilitating the study of variants; however, NGS is time-consuming and costly and not efficient during outbreaks, when rapid identification of VOCs is urgently needed. In such periods, there is a need for fast and accurate methods, such as real-time reverse transcription PCR in combination with probes, which can be used for monitoring and screening of the population for these variants. Thus, we developed a molecular beacon-based real-time RT-PCR assay according to the principles of spectral genotyping. This assay employs five molecular beacons that target ORF1a:ΔS3675/G3676/F3677, S:ΔH69/V70, S:ΔE156/F157, S:ΔΝ211, S:ins214EPE, and S:ΔL242/A243/L244, deletions and an insertion found in SARS-CoV-2 VOCs. This assay targets deletions/insertions because they inherently provide higher discrimination capacity. Here, the design process of the molecular beacon-based real-time RT-PCR assay for detection and discrimination of SARS-CoV-2 is presented, and experimental testing using SARS-CoV-2 VOC samples from reference strains (cultured virus) and clinical patient samples (nasopharyngeal samples), which have been previously classified using NGS, were evaluated. Based on the results, it was shown that all molecular beacons can be used under the same real-time RT-PCR conditions, consequently improving the time and cost efficiency of the assay. Furthermore, this assay was able to confirm the genotype of each of the tested samples from various VOCs, thereby constituting an accurate and reliable method for VOC detection and discrimination. Overall, this assay is a valuable tool that can be used for screening and monitoring the population for VOCs or other emerging variants, contributing to limiting their spread and protecting public health.

Aetiology of Community-Acquired Pneumonia and the Role of Genetic Host Factors in Hospitalized Patients in Cyprus.

Community-acquired pneumonia (CAP) remains the leading cause of hospitalization among infectious disease in Europe, and a major cause of morbidity and mortality. In order to determine and characterize the aetiology of CAP in hospitalized adults in Cyprus, respiratory and blood samples were obtained from hospitalized patients with CAP, and analyzed using Multiplex Real-Time PCR/RT-PCR, and ID/AMR enrichment panel (RPIP) analysis. Probe-based allelic discrimination was used to investigate genetic host factors in patients. The aetiology could be established in 87% of patients. The most prevalent viral pathogens detected were influenza A, SARS-CoV-2, and human rhinovirus. The most common bacterial pathogens detected were Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae. Antimicrobial resistance genes were identified in 23 patients. S. aureus was the most common AMR correlated strain in our study. A positive correlation was detected between bacterial infections and the NOS3 rs1799983 G allele and the FCGR2A rs1801274 G allele. A positive correlation was also detected between the TNF-α rs1800629 A allele and sepsis, while a negative correlation was detected with the ACE rs1799752 insertion genotype and the severity of pneumonia. In conclusion, the targeted NGS panel approach applied provides highly sensitive, comprehensive pathogen detection, in combination with antimicrobial resistance AMR insights that can guide treatment choices. In addition, several host factors have been identified that impact the disease progression and outcome.

A Preclinical Investigation on the Role of IgG Antibodies against Coagulant Components in Multiple Sclerosis.

The coagulation-inflammation interplay has recently been identified as a critical risk factor in the early onset of multiple sclerosis (MS), and antibodies against coagulation components have been recognized as contributing factors to thrombotic and inflammatory signaling pathways in diseases with overlapping symptoms to MS, paving the way for further research into their effects on MS pathology. The current study aimed to enlighten the role of IgG antibodies against coagulation components by performing a preclinical study, analyzing the astrocytic activation by purified IgG antibodies derived from 15 MS patients, and assessing their possible pro-inflammatory effects using a bead-based multiplexed immunoassay system. The results were compared with those obtained following astrocyte treatment with samples from 14 age- and gender-matched healthy donors, negative for IgG antibody presence. Serum samples collected from 167 MS patients and 40 age- and gender-matched controls were also analyzed for pro- and anti-inflammatory factors. According to our results, astrocytic activation in response to IgG treatment caused an upregulation of various pro-inflammatory factors, including cytokines, chemokines, and interleukins. Conversely, in serum samples from patients and controls, the pro-inflammatory factors did not differ significantly; medication may lower the levels in patients. Our findings suggest that antibodies may function as effectors in neuroinflammation and serve as targets for new treatments that eventually benefit novel therapeutic approaches.

Exposure to airborne SARS-CoV-2 in four hospital wards and ICUs of Cyprus. A detailed study accounting for day-to-day operations and aerosol generating procedures.

In any infectious disease, understanding the modes of transmission is key to selecting effective public health measures. In the case of COVID-19 spread, the strictness of the imposed measures outlined the lack of understanding on how SARS-CoV-2 transmits, particularly via airborne pathways. With the aim to characterize the transmission dynamics of airborne SARS-CoV-2, 165 and 62 air and environmental samples, respectively, were collected in four COVID-19 wards and ICUs in Cyprus and analyzed by RT-PCR. An alternative method for SARS-CoV-2 detection in air that provides comparable results but is less cumbersome and time demanding, is also proposed. Considering that all clinics employed 14 regenerations per hour of full fresh air inside patient rooms, it was hypothesized that the viral levels and the frequency of positive samples would be minimum outside of the rooms. However, it is shown that leaving the door opened in patient rooms hinders the efficiency of the ventilation system applied, allowing the virus to escape. As a result, the highest observed viral levels (135 copies m-3) were observed in the corridor of a ward and the frequency of positive samples in the same area was comparable to that inside a two-bed cohort. SARS-CoV-2 in that corridor was found primarily to lie in the coarse mode, at sizes between 1.8 and 10 µm. Similar to previous studies, the frequency of positive samples and viral levels were the lowest inside intensive care units. However, if a patient with sufficient viral load (Ct-value 31) underwent aerosol generating procedures, positive samples with viral levels below 45 copies m-3 were acquired within a 2 m distance of the patient. Our results suggest that a robust ventilation system can prevent unnecessary exposure to SARS-CoV-2 but with limitations related to foot traffic or the operations taking place at the time.