Influenza poses a serious threat to both individual and public health. This study aimed to investigate the virological and epidemiological characteristics of influenza infections and to explore the genetic diversity of the circulating influenza viruses. In total, 1886 nasopharyngeal specimens from patients with acute respiratory illnesses were tested against 13 respiratory viruses using a multiplex real-time PCR. Whole-genome sequencing, phylogenetic, and amino acid analyses of representative influenza strains were performed. At least one respiratory virus was detected in 869 (46.1%) patients; 87 (4.6%) were co-infected with two or three viruses. Influenza A(H1N1)pdm09 was the most prevalent virus (16.1%), followed by rhinoviruses (8.1%) and RSV (6.7%). Hemagglutinin (HA) genes of the 74 influenza A(H1N1)pdm09 viruses were categorized in subclades C.1.8, C.1.9, and C.1 within clade 5a.2a and D1, D.2, and D.3 within clade 5a.2a.1. The A(H3N2) viruses analyzed belonged to clade 2a.3a.1, subclades J.2 and J.1. The sequenced B/Victoria lineage viruses fell into clade V1A.3a.2, subclades C.5.6 and C.5.7. Amino acid substitutions in most viral proteins were identified compared with the vaccine strains, including in the HA antigenic sites. This study demonstrated the dominant distribution of the influenza A(H1N1)pdm09 virus among the respiratory viruses studied and the genetic diversity of the circulating influenza viruses.

Recently a resurgence of Streptococcus pyogenes infections has arisen, with concerns around the highly virulent M1_UK lineage. Our aim was to characterize S. pyogenes, the immune responses it causes, and to determine the presence of the M1_UK lineage in Sofia, Bulgaria. In our study, the infections were confirmed by culture testing or rapid antigen test. Identification was performed by MALDI-TOF and was followed up by antibiotic susceptibility testing (EUCAST). Virulence factors were identified using multiplex PCR and whole genome sequencing (WGS). Immune responses were measured through detection of serum complement levels, lymphocyte subsets, and cytokine profiling. Out of 82 children, 38 had scarlet fever and the rest had streptococcal pharyngitis. Strains were susceptible to penicillin (β-lactams), macrolides, clindamycin, tetracyclines, co-trimoxazole, fluoroquinolones, and linezolid. Superantigen profiles were identified: SpeA + SpeJ (45%), SpeC, and SpeI + SpeH (27.5% each). A novel Multilocus sequence typing (MLST) haplotype in the mutS gene (d90b) was found in four strains. The M1_UK lineage was detected for the first time in Bulgaria. We observed an increase in complement fractions C3 and C4 and a decrease in T lymphocytes. A significant increase in the levels of IFN-γ, IL-6, and IL-10 with corresponding reduction in IL-17A were revealed. In conclusion, the studied S. pyogenes strains were characterized by their susceptibility to antibiotics and the predominance of SpeA superantigen; for the first time in Bulgaria the presence of M1_UK and a novel SNP variation in the mutS gene (d90b) were found. A mixed pattern of pro- and anti-inflammatory immune responses in patients was observed.

Studies of the COVID-19 pandemic revealed differences in the clinical course between paediatric and adult patients. In-depth research on whether T follicular cells support these observations is still ongoing. Circulating Tfh and Tfr cells were examined to identify specific immune responses in paediatric and adult individuals who recovered from asymptomatic to mild SARS-CoV-2 infection. Circulating Tfh and Tfr cells were evaluated by flow cytometry. To evaluate specific anti-SARS-CoV-2 immune responses, anti-Spike RBD IgG antibodies were measured by an enzyme-linked fluorescent assay, anti-Spike neutralizing activity - by an in vitro cell-based assay, and IFN-γ T cell responses were evaluated via ELISpot assay. Age-specific differences in the profiles of the CXCR5⁺CD4⁺ T cells that distinguish them from CXCR5- counterparts were detected. An increased percentage of cTfr cells and decreased percentage of cTfh cells was observed in the paediatric group, which resulted in a decreased cTfh/cTfr ratio. Increased proportions of ICOS- and HLA-DR-expressing cTfh and cTfr cells were also observed, consistent with the high proportion of antibody-responders among children. In contrast, a greater percentage of adults than children exhibited IFN-γ-positive T cells in response to the four viral peptides. In adult responders, a good correlation was found between the presence of plasmablasts and the cTfh/cTfr ratio. Antibody neutralizing capacity did not differ between children and adults. Circulating Tfh and Tfr cells exhibit specific age-dependent differences, which may contribute to the immunologic background of the clinical symptoms of SARS-CoV-2 infection.

Streptococcus pyogenes, Group A Streptococcus (GAS), is the most common cause of bacterial tonsillitis, scarlet fever, impetigo, etc. in children and adults. Innate and adaptive host immune responses are fundamental for human defence against GAS, but are also important to the clinical manifestations of the infection. Our aim was to study the humoral and cellular immune responses in children infected with GAS and diagnosed with tonsillitis in the Clinic of Pediatrics in University Hospital “Lozenets”. The study included 22 infected children and 20 healthy controls. The following immune parameters were investigated: i) Humoral immune response – total serum IgG, IgM, IgA; C3, C4 – complement components, as well as the serum cytokines IFNγ, IL-6, IL-1β, TNFα, IL-17A, IL-10. ii) Cellular immune subsets were tested by immunophenotyping of total T cells, B cells, NK cells, T helper and T cytotoxic subsets. In this cross-sectional study we found a significant increase in the complement components C3 and C4, increased production of serum IFNγ, IL-6 and IL-10 and decreased levels of IL-17A in infected children with tonsillitis, compared to healthy controls. The cellular immune parameters in infected children showed lymphopenia, reduced total T lymphocytes and T-helper subpopulation, compared to healthy controls. We assumed that these imbalanced immune responses, found in infected children, are connected with GAS infection and deserved more detailed investigation.

Influenza poses a serious threat to both individual and public health . This study aimed to investigate the virological and epidemiological characteristics of influenza infections and to explore the genetic diversity of the circulating influenza viruses. In total, 1886 nasopharyngeal specimens from patients with acute respiratory illnesses were tested against 13 respiratory viruses using a multiplex real-time PCR. Whole-genome sequencing, phylogenetic, and amino acid analyses of representative influenza strains were performed. At least one respiratory virus was detected in 869 (46.1%) patients, 87 (4.6%) were co-infected with two or three viruses. Influenza A(H1N1)pdm09 was the most prevalent virus (16.1%), followed by rhinoviruses (8.1%) and RSV (6.7%). Hemagglutinin (HA) genes of the 74 influenza A(H1N1)pdm09 viruses were categorized in subclades C.1.8, C.1.9, and C.1 within clade 5a.2a and D1, D.2, and D.3 within clade 5a.2a.1 The A(H3N2) viruses analyzed belonged to clade 2a.3a.1, subclades J.2 and J.1. The sequenced B/Victoria lineage viruses fell into clade V1A.3a.2, subclades C.5.6 and C.5.7. Amino acid substitutions in most viral proteins were identified compared with the vaccine strains, including in the HA antigenic sites. This study demonstrated the dominant distribution of the influenza A(H1N1)pdm09 virus among the respiratory viruses studied and the genetic diversity of the circulating influenza viruses.