Undetected Omicron Transmission in Romania-Report of the First Detected Case of Locally Acquired Omicron Infection and Complete Epidemiological Investigation.

The occurrence of the omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has importantly impacted surveillance and diagnosis, and has changed the therapeutic landscape of coronavirus disease 2019 (COVID-19). We present the first documented case of locally acquired SARS-CoV-2 omicron variant in Romania in a patient with no recent travel outside the country. We also present the full results of the epidemiological investigation that led to the identification of the index case in a co-worker who had developed mild symptoms shortly after returning from the UK and who had undergone multiple rapid antigen tests with negative results prior to being tested by RT-PCR. We highlight potential lessons learned and describe further directions for actionable research and development in the field of COVID-19.

Clinical and molecular epidemiology of influenza viruses from Romanian patients hospitalized during the 2019/20 season.

Two main mechanisms contribute to the continuous evolution of influenza viruses: accumulation of mutations in the hemagglutinin and neuraminidase genes (antigenic drift) and genetic re-assortments (antigenic shift). Epidemiological surveillance is important in identifying new genetic variants of influenza viruses with potentially increased pathogenicity and transmissibility. In order to characterize the 2019/20 influenza epidemic in Romania, 1042 respiratory samples were collected from consecutive patients hospitalized with acute respiratory infections in the National Institute for Infectious Diseases "Prof. Dr. Matei Balș", Bucharest Romania and tested for influenza A virus, influenza B virus and respiratory syncytial virus (RSV) by real-time PCR. Out of them, 516 cases were positive for influenza, with relatively equal distribution of influenza A and B. Two patients had influenza A and B co-infection and 8 patients had influenza-RSV co-infection. The most severe cases, requiring supplemental oxygen administration or intensive care, and the most deaths were reported in patients aged 65 years and over. Subtyping showed the predominance of A(H3N2) compared to A(H1N1)pdm09 pdm09 (60.4% and 39.6% of all subtyped influenza A isolates, respectively), and the circulation of Victoria B lineage only. Influenza B started to circulate first (week 47/2019), with influenza A appearing slightly later (week 50/2019), followed by continued co-circulation of A and B viruses throughout the season. Sixty-eight samples, selected to cover the entire influenza season and all circulating viral types, were analysed by next generation sequencing (NGS). All A(H1N1)pdm09 sequences identified during this season in Romania were clustered in the 6b1.A clade (sub-clades: 6b1.A.183P -5a and 6b1.A.187A). For most A(H1N1)pdm09 sequences, the dominant epitope was Sb (pepitope = 0.25), reducing the vaccine efficacy by approximately 60%. According to phylogenetic analysis, influenza A(H3N2) strains circulating in this season belonged predominantly to clade 3C.3A, with only few sequences in clade 3C.2A1b. These 3C.2A1b sequences, two of which belonged to vaccinated patients, harbored mutations in antigenic sites leading to potential reduction of vaccine efficacy. Phylogenetic analysis of influenza B, lineage Victoria, sequences showed that the circulating strains belonged to clade V1A3. As compared to the other viral types, fewer mutations were observed in B/Victoria strains, with limited impact on vaccine efficiency based on estimations.

The Predictive Value of Mutation Screening for Anticipating COVID-19 Waves.

Emerging SARS-CoV-2 strains continue to generate difficulties for authorities and health care professionals worldwide due to enhanced transmissibility and/or immune response evasion. The appearance of the Alpha and Delta strains has been associated with substantial increases in the number of COVID-19 cases and associated deaths. Whole Genome Sequencing (WGS) continues to be the gold standard for molecular surveillance of the pandemics but other assays such as mutation genotyping can be used to reduce costs and allocated time. This study investigates the efficiency of mutation screening tests compared to WGS and their predictive value to anticipate future waves. A very high degree of fidelity for this type of assay was found, regardless of the method used. The positive predictive value (PPV) of 4/5 markers was over 95% for the detection of Alpha and Delta variants. By estimating the prevalence of the Alpha and Delta strains using genotyping assays and fitting the data to a mathematical model, a five week period between the point of exponential growth of variant prevalence and a drastic increase in case numbers was found. For that reason, raising awareness about the efficacy of mutation screening could help authorities adopt better measures in the future.

Exhaustion and senescence of CD4 and CD8 T cells that express co-stimulatory molecules CD27 and CD28 in subjects that acquired HIV by drug use or by sexual route.

INTRODUCTION: The human immunodeficiency virus (HIV) infection leads to immune activation, senescence and exhaustion of T cells. Co-stimulatory molecules play important roles in controlling these processes. The CD28 signaling triggers efficient T cell activation, while CD27 provides survival signals to CD28- T cells. Loss of these molecules was associated with senescent phenotype and resistance to checkpoint inhibitors.Romania has faced an HIV outbreak among people who inject drugs (PWID), most of them chronically infected with hepatitis C virus (HCV). HIV/HCV co-infection was associated with increased immune activation and rapid disease progression. METHODS: We evaluated by flow cytometry the expression of CD27, CD28, CD38, HLA-DR, CD57 and PD-1 on CD4 and CD8 T cells from 34 subjected infected with HIV (22 PWID and 12 people who acquired HIV by sexual route - PWHS) and 18 HIV-negative individuals (controls). RESULTS: We found that as compared to controls, HIV patients, regardless of infection route, have high percentages of intermediately differentiated (CD27+CD28-) and low percentages of less differentiated (CD27+CD28+) CD8 T cells. Significantly higher levels of CD8+CD27+CD28- T cells were found in PWHS than in PWID. A lower percentage of intermediately and highly differentiated (CD27-CD28-) CD8 T cells express CD57 in people living with HIV (PLWH) than in controls. Increased levels of less and intermediately differentiated CD4 and CD8 T cells expressing PD-1 were identified in PLWH, especially in PWID; these directly correlated with HIV viral load and T cell activation and negatively correlated with CD4 counts. CONCLUSIONS: Our data show that induction of PD-1 on T cells expressing co-stimulatory molecules CD27 and/or CD28 might contribute to poor control of HIV infection and to immune activation.

Short Communication:Evidence of Novel SARS-CoV-2 Variants Circulation in Romania.

New SARS-CoV-2 variants are constantly emerging and putting a strain on public health systems by spreading faster and potentially evading immune protection through vaccination. One of these strains is the B.1.1.7 variant that has initially been described in the United Kingdom and has subsequently spread to several countries. Monitoring the amplification of the S gene-a major hotspot for molecular evolution-by reverse transcription polymerase chain reaction (RT-PCR) allows rapidly screening for such variants. This report describes the detection of sequence variants in Romania by using this strategy followed by next-generation sequencing of the entire genome for confirmation and further characterization. One B.1.1.7 and three B.1.258 sequences were confirmed. Each of these strains presented additional mutations with possible impact on the replicative capacity. Public health strategies should be devised to ensure molecular monitoring of SARS-CoV-2 evolution during the pandemic and allow adequate and rapid reaction.

Molecular Epidemiology Analysis of SARS-CoV-2 Strains Circulating in Romania during the First Months of the Pandemic.

BACKGROUND: The spread of SARS-CoV-2 generated an unprecedented global public health crisis. Soon after Asia, Europe was seriously affected. Many countries, including Romania, adopted lockdown measures to limit the outbreak. AIM: We performed a molecular epidemiology analysis of SARS-CoV-2 viral strains circulating in Romania during the first two months of the epidemic in order to detect mutation profiles and phylogenetic relatedness. METHODS: Respiratory samples were directly used for shotgun sequencing. RESULTS: All Romanian sequences belonged to lineage B, with a different subtype distribution between northern and southern regions (subtype B.1.5 and B.1.1). Phylogenetic analysis suggested that the Romanian epidemic started with multiple introduction events from other European countries followed by local transmission. Phylogenetic links between northern Romania and Spain, Austria, Scotland and Russia were observed, as well as between southern Romania and Switzerland, Italy, France and Turkey. One viral strain presented a previously unreported mutation in the Nsp2 gene, namely K489E. Epidemiologically-defined clusters displayed specific mutations, suggesting molecular signatures for strains coming from areas that were isolated during the lockdown. CONCLUSIONS: Romanian epidemic was initiated by multiple introductions from European countries followed by local transmissions. Different subtype distribution between northern and southern Romania was observed after two months of the pandemic.

NGS combined with phylogenetic analysis to detect HIV-1 dual infection in Romanian people who inject drugs.

Dual HIV infections are possible and likely in people who inject drugs (PWID). Thirty-eight newly diagnosed patients, 19 PWID and 19 heterosexually HIV infected were analyzed. V2V3 loop of HIV-1 env gene was sequenced on the NGS platform 454 GSJunior (Roche). HIV-1 dual/multiple infections were identified in five PWID. For three of these patients, the reconstructed variants belonged to pure F1 subtype and CRF14_BG strains according to phylogenetic analysis. New recombinant forms between these parental strains were identified in two PWID samples. NGS data can provide, with the help of phylogenetic analysis, important insights about the intra-host sub-population structure.

Epidemic dispersion of HIV and HCV in a population of co-infected Romanian injecting drug users.

Co-infections with HIV and HCV are very frequent among people who inject drugs (PWID). However, very few studies comparatively reconstructed the transmission patterns of both viruses in the same population. We have recruited 117 co-infected PWID during a recent HIV outbreak in Romania. Phylogenetic analyses were performed on HIV and HCV sequences in order to characterize and compare transmission dynamics of the two viruses. Three large HIV clusters (2 subtype F1 and one CRF14_BG) and thirteen smaller HCV transmission networks (genotypes 1a, 1b, 3a, 4a and 4d) were identified. Eighty (65%) patients were both in HIV and HCV transmission chains and 70 of those shared the same HIV and HCV cluster with at least one other patient. Molecular clock analysis indicated that all identified HIV clusters originated around 2006, while the origin of the different HCV clusters ranged between 1980 (genotype 1b) and 2011 (genotypes 3a and 4d). HCV infection preceded HIV infection in 80.3% of cases. Coincidental transmission of HIV and HCV was estimated to be rather low (19.65%) and associated with an outbreak among PWID during detention in the same penitentiary. This study has reconstructed and compared the dispersion of these two viruses in a PWID population.

Dysregulation of anergy-related factors involved in regulatory T cells defects in Systemic Lupus Erythematosus patients: Rapamycin and Vitamin D efficacy in restoring regulatory T cells.

AIM: Systemic Lupus Erythematosus (SLE) patients display dysfunctions in T cell activation and anergy. Therefore the aims of our study were to explore the expression of anergy-related factors in CD4+ T cells in relationship with regulatory T cells (Tregs) frequency in SLE patients and to identify strategies to redress these defects. METHOD: Casitas B-cell lymphoma b (Cbl-b) and 'gene related to anergy in lymphocytes' (GRAIL) proteins were analyzed in peripheral blood mononuclear cells (PBMCs) from SLE patients and healthy donors (HD) by immunoblotting. cbl-b, grail, growth response factors (egr)2 and egr3 messenger RNAs (mRNAs) were evaluated by real-time polymerase chain reaction in SLE and HD PBMCs and CD4+ T cells. Phenotypic and functional characterization of CD4+ T cells was performed by flow cytometry. Tregs expansion protocol consisted in culturing CD4+ T cells for 14 or 21 days of experimental activation with anti-CD3 and anti-CD28 monoclonal antibodies, human recombinant interleukin (hrIL)-2, in the absence or presence of rapamycin (Rapa) or 1,25-(OH)2D3 (vitamin D: VitD). RESULTS: SLE PBMCs expressed low levels of Cbl-b and GRAIL proteins. Both SLE PBMCs and CD4+ T cells expressed low levels of egr2/3 mRNAs. SLE patients had a reduced number of Tregs with impaired suppressive activity. An association between egr2 mRNA level in CD4+ T cells and Tregs percentage was identified. Experimental activation of CD4+ T cells in the presence of hrIL-2 and Rapa or VitD induced the expansion of SLE Tregs. However, on long-term, only Rapa exposure of SLE CD4+ T cells yielded high numbers of Tregs with sustained suppressive activity. CONCLUSION: Our results suggest a new strategy to correct defects in CD4+ T cell tolerance mechanisms that may prove beneficial in SLE.

Recent HIV-1 Outbreak Among Intravenous Drug Users in Romania: Evidence for Cocirculation of CRF14_BG and Subtype F1 Strains.

Since 2011, Romania has faced an HIV outbreak among injecting drug users (IDUs). Our aim was to identify and describe clinical and epidemiological patterns of this outbreak. A cross-sectional study enrolled 138 IDUs diagnosed with HIV infection between 2011 and 2013 with 58 sexually infected individuals included as the control group. The IDUs had a long history of heroin abuse (10 years) and a recent history of new psychostimulant injection (3-4 years). Classical epidemiological data and molecular techniques were used to describe the transmission dynamics. A high prevalence of hepatitis C virus (HCV) coinfection was noted (98.6%) compared to the control group (10.3%) (p<0.001). IDUs had initially been infected with HCV. HIV infection was more recent, linked to starting injecting stimulants. HIV subtype analysis showed a predominance of the local F1 strain in both IDUs and sexually infected patients; in IDUs it also identified 28 CRF14_BG recombinants and six unique recombinant forms (URFs) between F1 and CRF14_BG. A few patients from both risk groups were infected with subtype B. Among IDUs, CRF14_BG was associated with a lower CD4 cell count and more advanced stages of disease, which correlated with CXCR4 tropism. Phylogenetic analysis revealed the spread of HIV through three major IDU clusters of recent date. Among IDUs with CRF14_BG, some reported travel abroad (Spain, Greece). By identifying clusters of IDUs with related viruses, molecular epidemiologic methods provide valuable information on patterns of HIV transmission that can be useful in planning appropriate harm reduction interventions.

Peripheral blood lymphocytes analysis detects CD100/SEMA4D alteration in systemic sclerosis patients.

It was suggested that the immune system plays an important role at least in the amplification of the main elements in systemic sclerosis (SSc), an autoimmune disease with an incompletely elucidated pathogenesis. Elucidation of the mechanisms involved in the interaction between T and B cells, major players of the immune system, could contribute to a better understanding of some of clinical and pathological manifestations of SSc. Recently, abnormalities in Semaphorin 4D (Sema4D/CD100) or CD72, two contrareceptors involved in T and B cells cooperation, were associated with autoimmunity. Therefore, we investigated CD100 and CD72 expression level on T and B cells in attempting to establish their role in SSc pathogenesis. The results revealed augmented percentages of CD100(high) T and B cells, significantly increased expression of CD100 on CD4(+) T cells and frequently detectable levels of soluble CD100 in SSc patient sera compared to healthy donors. In SSc, CD100 dysregulations were associated with anti-Scl70 antibodies production, disease type, thickening of skin, disease duration, or with active inflammation processes. In consequence, dysregulations in CD100 expression and release could play a role in SSc development and/or maintenance.

Antibody and splenocyte proliferation response to whole inactivated Streptococcus pneumoniae serotype 1, 3 and 6B in mice.

Animal models of infection and protection on the topic of the Streptococcus pneumoniae (S. pneumoniae) have encountered many difficulties generated by low immunogenicity, a characteristic of polysaccharide capsular bacteria and difference of virulence between serotypes and strains. We have explored the immune response after immunization with heat inactivated S. pneumoniae serotype 1, 3 and 6B in C57BL/6 mice by IgM and IgG detection, and by splenocyte in vitro 5-ethynyl-2'-deoxyuridine (EdU) incorporation after antigen specific stimulation, as a proposed method of cellular immune response evaluation. Antibody titer persistence after immunization was not lengthy while antigen specific proliferation response detected by EdU assay was remnant. Intraperitoneal (i.p.) challenge with serotype 6B S. pneumoniae proved that antibody titers and the detected specific cellular immune response do not cover seroprotective necessity and do not confer improved immunologic memory in comparison to non-immunized mice, which show natural resistance.

Role of cellular immunity in systemic sclerosis pathogenesis: update on CD4+T cells population studies.

Immunologic abnormalities observed in Systemic Sclerosis (SSc) patients consist of chronic mononuclear cell infiltration of affected tissues, dysregulation of lymphokine and growth factor production, and autoantibodies production. Expansion of CD4+T cells within the tissue seems to involve their activation that precedes this process. Therefore, CD4+T cells activation, as an early immune event, appears to be an important process in the development and maintaining of SSc. In SSc the disturbance of peripheral tolerance mechanisms could be also responsible for CD4+T cells activation. Consequently, we reevaluated CD4+T cells positive for CD25, GITR, CTLA-4, CD45RO, or Foxp3 in SSc patients, by comparison with healthy donors (HDs), and in correlation with clinical features of the disease. Our results reargued for activation of peripheral blood CD4+T cells in SSc patients. Thus, increased percentages of CD25+ and GITR+ CD4+T cells were found in SSc patients by comparison with HDs. Direct correlation between the percentage of GITR+CD4+T cells and disease activity recommended these cells as a good candidate for disease progression. In SSc patients, the negative regulators of T cells activation are also affected. Thus, CTLA-4+ and Foxp3+ CD4+T cell percentages were significantly reduced in SSc patients when compared to HDs. Indirect correlation between the percentage of CD152+CD4+T cells and autoantibodies (aScl70) presence or disease type highlighted the role of these cells in the disturbance of peripheral tolerance. The absence of the direct correlation between CD152+CD4+T cells and CD45RO+CD4+T cells, correlation observed only in HDs, raised the hypothesis that in SSc patients, memory T cells can be easily activated, and by consequence, they can enter within affected tissues. These data reconfirm the activation state of SSc CD4+T cells and point out some abnormalities in peripheral tolerance mechanisms that can contribute to SSc pathogeny.

Quantification and molecular characterization of regulatory T cells in connective tissue diseases.

The aim of our study was to investigate and characterize regulatory T cells (Treg) in peripheral blood of patients with connective tissue diseases (Systemic lupus erythematosus, systemic sclerosis, Sjögren's syndrome, poly- and dermatomyositis) as compared with blood from healthy controls. Treg cells were quantified and phenotypically characterized by flow cytometry while the expression level of Foxp3 mRNA was evaluated by real time PCR. A reduced percentage of peripheral blood Treg cells was found in patients than in controls, irrespective of the type of connective tissue disease. Treg cells, especially those expressing one of the phenotypical markers, seemed to differ not only between patients and healthy controls but also among types of diseases. Additionally, the presence of autoantibodies as well as disease activity appeared to be correlated with particular Treg cell populations, especially those expressing one of the examined phenotypical markers. Correlations with therapy suggested that glucocorticoids plus antimalarial or other immunosuppressor drugs diminished the percentage of Treg cells, especially of those with memory phenotype. These findings indicated dysregulations at the level of Treg cells and suggested an involvement of these cells in the pathology of connective tissue diseases. Moreover, our data are in agreement with the suggestion that Treg cells could be therapeutic targets for some autoimmune diseases.

PI3K/Akt signaling in peripheral T lymphocytes from systemic lupus erythematosus patients.

PI3K/Akt/mTOR signaling pathway plays an important role in cellular proliferation and growth signaling. It was demonstrated that murine models presenting activated PI3K/Akt/mTOR signaling pathway in lymphocytes develop features of systemic autoimmunity, linking this pathway to autoimmune diseases. Therefore, the goal of our study was to analyze this signaling axis in Systemic Lupus Erythematosus (SLE), the prototype of systemic autoimmune diseases, focusing on Akt and p70S6k, two components of this pathway. Our results demonstrated that both expression and phosphorylation levels of Akt are more increased in SLE than in healthy donors (HDs) CD4+ T cells suggesting an up-regulation of PI3K and mTOR activities. This result was also suggested when p70S6k, one of mTOR substrate, was evaluated. Indeed, in SLE CD4+ T cells an enhancement of p70S6k activity, in direct correlation with its expression level, was found. Since p27kip1, an inhibitor of cell cycle progression, is one of the Akt substrates, we analyzed its expression level in relationship with cell cycle progression and apoptosis. The results demonstrated that p27kip1 expression level was significantly decreased in SLE than in HDs CD4+ T cells. In SLE p27kip1 level was inversely correlated with the percentage of peripheral lymphocytes in apoptosis and in S phase of the cell cycle. Therefore, the increased activity of PI3K/Akt/mTOR signaling pathway and, as a result, the drop of p27kip1 levels observed in CD4+ T cells isolated from SLE patients might explain the accumulation of SLE lymphocytes in S and G2/M cell cycle phases where they undergo apoptosis.