Genomic Characterization of 16S rRNA Methyltransferase-Producing Enterobacterales Reveals the Emergence of Klebsiella pneumoniae ST6260 Harboring rmtF, rmtB, blaNDM-5, blaOXA-232 and blaSFO-1 Genes in a Cancer Hospital in Bulgaria.

Background: Acquired 16S rRNA methyltransferases (16S-RMTases) confer high-level resistance to aminoglycosides and are often associated with ß-lactam and quinolone resistance determinants. Methods: Using PCR, whole-genome sequencing and conjugation experiments, we conducted a retrospective genomic surveillance study of 16S-RMTase-producing Enterobacterales, collected between 2006 and 2023, to explore transmission dynamics of methyltransferase and associated antibiotic resistance genes. Results: Among the 10,731 consecutive isolates, 150 (1.4%) from 13 species carried armA (92.7%), rmtB (4.7%), and rmtF + rmtB (2.7%) methyltransferase genes. The coexistence of extended-spectrum ß-lactamase (blaCTX-M-3/15, blaSHV-12, blaSFO-1), carbapenemase (blaNDM-1/5, blaVIM-1/4/86, blaOXA-48), acquired AmpC (blaCMY-2/4/99, blaDHA-1, blaAAC-1), and plasmid-mediated quinolone resistance (qnrB, qnrS, aac(6')-Ib-cr) genes within these isolates was also detected. Methyltransferase genes were carried by different plasmids (IncL/M, IncA/C, IncR, IncFIB, and IncFII), suggesting diverse origins and sources of acquisition. armA was co-transferred with blaCTX-M-3/15, blaNDM-1, blaVIM-4/86, blaOXA-48, blaCMY-4, aac(6')-Ib-cr, qnrB, and qnrS, while rmtF1 was co-transferred with blaSFO-1, highlighting the multidrug-resistant nature of these plasmids. Long-read sequencing of ST6260 K. pneumoniae isolates revealed a novel resistance association, with rmtB1 and blaNDM-5 on the chromosome, blaOXA-232 on a conjugative ColKP3 plasmid, and rmtF1 with blaSFO-1 on self-transmissible IncFIB and IncFII plasmids. Conclusions: The genetic plasticity of plasmids carrying methyltransferase genes suggests their potential to acquire additional resistance genes, turning 16S-RMTase-producing Enterobacterales into a persistent public health threat.

Bacterial and Viral Co-Infections in COVID-19 Patients: Etiology and Clinical Impact.

BACKGROUND: Mixed infections can worsen disease symptoms. This study investigated the impact of mixed infections with viral and bacterial pathogens in patients positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: Using the in-house multiplex PCR method, we tested 337 SARS-CoV-2 positive samples for co-infections with three bacterial and 14 other viral pathogens. RESULTS: Between August 2021 and May 2022, 8% of 337 SARS-CoV-2-positive patients had bacterial co-infections, 5.6% had viral co-infections, and 1.4% had triple mixed infections. The most common causes of mixed infections were Haemophilus influenzae (5.93%) and respiratory syncytial virus (RSV) (1.18%). Children < 5 years old had more frequent co-infections than adults < 65 years old (20.8% vs. 16.4%), while adults showed a more severe clinical picture with a higher C-reactive protein (CRP) level (78.1 vs.16.2 mg/L; p = 0.033), a lower oxygen saturation (SpO2) (89.5 vs. 93.2%), and a longer hospital stay (8.1 vs. 3.1 days; p = 0.025) (mean levels). The risk of a fatal outcome was 41% in unvaccinated patients (p = 0.713), which increased by 2.66% with co-infection with two pathogens (p = 0.342) and by 26% with three pathogens (p = 0.005). Additionally, 50% of intensive care unit (ICU) patients had a triple infection, compared with only 1.3% in the inpatient unit (p = 0.0029). The risk of death and/or ICU admission was 12 times higher (p = 0.042) with an additional pathogen and increased by 95% (p = 0.003) with a third concomitant pathogen. CONCLUSIONS: Regular multiplex testing is important for prompt treatment and targeted antibiotic use.

Prevalence of Toscana Virus Antibodies in Residents of Bulgaria: A Nationwide Study Following the Pandemic of COVID-19.

Introduction: Toscana virus (TOSV) is an arthropod-borne virus transmitted by sandflies and is mainly found in countries around the Mediterranean basin. In this article, we present the findings of a seroprevalence study on TOSV in Bulgaria. We aim to assess the current epidemiological situation regarding TOSV in the country and raise clinical awareness. Methods: Serum samples (n = 1892) were collected in December 2023. Serological tests were performed using a commercial anti-TOSV ELISA kit. Results: Specific immunoglobulin G (IgG) antibodies were detected in 6.4% (121/1892) of the participants. A significantly higher seropositivity rate was found in the age group over 65 years compared with the age groups 18-29 and 40-64 (11.8% vs. 3.9% vs. 3.4%), as well as in males compared with females (8.0% vs. 5.3%). The seroprevalence rates in districts ranged from 0% to 18.5%. Higher seropositivity was found in the southern and northern regions. Conclusions: The seroprevalence rate of TOSV in Bulgaria, found in this study, is a significant decrease compared with the seropositivity rate of 24.5% reported in the country in 2018. The reasons for this are unknown and could possibly be related to the COVID-19 pandemic and the constantly changing environmental conditions. There is also a possibility that the higher seropositivity detected in 2018 together with the rise in clinical cases reported from endemic countries around that time might have been due to an unrecognized TOSV outbreak taking place in this period. Continued clinical awareness and surveillance are necessary for recognition and management of potential cases of TOSV neuroinfection, especially during summer.

Serological Assessment of Lyme borreliosis in Bulgaria: A Nationwide Study.

Lyme borreliosis (LB), a tick-borne infection caused by bacteria in the Borrelia burgdorferi sensu lato complex, is increasingly prevalent on the Balkan Peninsula, including Bulgaria, where it is the most common tick-borne disease. This study aimed to assess the seroprevalence of LB across Bulgaria by analyzing 1892 serum samples for specific IgG antibodies using a two-tier testing protocol involving an ELISA and immunoblot methods. The results revealed an overall seroprevalence rate of 5.4%, with significant variation based on age, sex, and residence. Seroprevalence increased with age, peaking at 8.4% in individuals over 65 years. Males had a seroprevalence of 8.4% compared to 3.3% in females, and rural residents showed higher seroprevalence (10.2%) compared to urban residents (4.4%). Regional analysis indicated that seroprevalence ranged from 0.0% to 20.0%, with higher rates in northern provinces such as Gabrovo (18.9%) and Targovishte (20.0%). This study highlights the importance of two-step testing protocols for accurate diagnosis and underscores the need for increased awareness and further research to enhance public health measures and the management of LB in Bulgaria.

Monkeypox in Bulgaria: Significance of Various Clinical Samples, Clinical Manifestation, and Molecular Detection.

Background/Objectives: Monkeypox (mpox) is currently the most common orthopoxvirus (OPXV) zoonotic disease, and, since 2022, there has been atypical person-to-person transmission observed in non-endemic countries. The present study aimed to investigate the frequency of monkeypox virus (MPXV) and OPXV DNA detection in recommended and alternative clinical materials taken during the acute and convalescent phases of infection in Bulgarian patients. Methods: The study included laboratory investigation by real time PCR of 181 clinical samples from 42 Bulgarian patients with possible mpox infections. Results: MPXV DNA was detected in 23/181 (12.71%), and OPXV DNA in 20/181 (11.05%) clinical samples. There were six mpox-confirmed patients aged 23 to 44. At the highest frequency, MPXV and OPXV DNA were detected in samples of vesicular contents (6/6) and nasal/oropharyngeal secretions (5/6 and 4/6) during the first three days from the appearance of clinical symptoms. We demonstrated MPXV and OPXV DNA in alternative samples (urine, feces, ejaculate, and saliva), and in follow-up patient samples, taken two weeks after mpox confirmation in the convalescent phase (vesicular contentsand urine). Conclusions: Our findings suggested that MPXV may be detected in a larger set of clinical materials, including alternatives, where the virus can persist for more than two weeks.

Resurgence of influenza with increased genetic diversity of circulating viruses during the 2022-2023 season.

Introduction. After two seasons of absence and low circulation, influenza activity increased significantly in the winter of 2022-2023. This study aims to characterize virological and epidemiological aspects of influenza infection in Bulgaria during the 2022-2023 season and perform a phylogenetic/molecular analysis of the hemagglutinin (HA) and neuraminidase (NA) sequences of representative influenza strains.Hypothesis/Gap Statement. Influenza A and B viruses generate new genetic groups/clades each season, replacing previously circulating variants. This results in increased antigenic distances from current vaccine strains. Strengthening existing influenza surveillance is essential to meet the challenges posed by the co-circulation of influenza and SARS-CoV-2.Methodology. We tested 2713 clinical samples from patients with acute respiratory illnesses using a multiplex real-time RT-PCR kit (FluSC2) to detect influenza A/B and Severe acute respiratory syndrome coronavirus-2(SARS-CoV-2) simultaneously. Representative Bulgarian influenza strains were sequenced at the WHO Collaborating Centres in London, UK, and Atlanta, USA.Results. Influenza virus was detected in 694 (25.6 %) patients. Of these, 364 (52.4 %), 213 (30.7 %) and 117 (16.9 %) were positive for influenza A(H1N1)pdm09, A(H3N2) and B/Victoria lineage virus, respectively. HA genes of the 47 influenza A(H1N1)pdm09 viruses fell into clades 5a.2. and 5a.2a.1 within the 6B.5A.1A.5a.2 group. Twenty-seven A(H3N2) viruses belonging to subclades 2b, 2a.1, 2a.1b and 2a.3a.1 within the 3C.2a1b.2a.2 group were analysed. All 23 sequenced B/Victoria lineage viruses were classified into the V1A.3a.2 group. We identified amino acid substitutions in HA and NA compared with the vaccine strains, including several substitutions in the HA antigenic sites.Conclusion. The study's findings showed genetic diversity among the influenza A viruses and, to a lesser extent, among B viruses, circulating in the first season after the lifting of anti-COVID-19 measures.

Continued circulation of mpox: an epidemiological and phylogenetic assessment, European Region, 2023 to 2024.

During the summer of 2023, the European Region experienced a limited resurgence of mpox cases following the substantial outbreak in 2022. This increase was characterised by asynchronous and bimodal increases, with countries experiencing peaks at different times. The demographic profile of cases during the resurgence was largely consistent with those reported previously. All available sequences from the European Region belonged to clade IIb. Sustained efforts are crucial to control and eventually eliminate mpox in the European Region.

Epidemiological and Genetic Characteristics of Respiratory Viral Coinfections with Different Variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

This study aimed to determine the incidence and etiological, seasonal, and genetic characteristics of respiratory viral coinfections involving severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Between October 2020 and January 2024, nasopharyngeal samples were collected from 2277 SARS-CoV-2-positive patients. Two multiplex approaches were used to detect and sequence SARS-CoV-2, influenza A/B viruses, and other seasonal respiratory viruses: multiplex real-time polymerase chain reaction (PCR) and multiplex next-generation sequencing. Coinfections of SARS-CoV-2 with other respiratory viruses were detected in 164 (7.2%) patients. The most common co-infecting virus was respiratory syncytial virus (RSV) (38 cases, 1.7%), followed by bocavirus (BoV) (1.2%) and rhinovirus (RV) (1.1%). Patients ≤ 16 years of age had the highest rate (15%) of mixed infections. Whole-genome sequencing produced 19 complete genomes of seasonal respiratory viral co-pathogens, which were subjected to phylogenetic and amino acid analyses. The detected influenza viruses were classified into the genetic groups 6B.1A.5a.2a and 6B.1A.5a.2a.1 for A(H1N1)pdm09, 3C.2a1b.2a.2a.1 and 3C.2a.2b for A(H3N2), and V1A.3a.2 for the B/Victoria lineage. The RSV-B sequences belonged to the genetic group GB5.0.5a, with HAdV-C belonging to type 1, BoV to genotype VP1, and PIV3 to lineage 1a(i). Multiple amino acid substitutions were identified, including at the antibody-binding sites. This study provides insights into respiratory viral coinfections involving SARS-CoV-2 and reinforces the importance of genetic characterization of co-pathogens in the development of therapeutic and preventive strategies.

Genomic Characterization of Carbapenemase-Producing Enterobacter hormaechei, Serratia marcescens, Citrobacter freundii, Providencia stuartii, and Morganella morganii Clinical Isolates from Bulgaria.

Carbapenemase-producing Enterobacter spp. Serratia marcescens, Citrobacter freundii, Providencia spp., and Morganella morganii (CP-ESCPM) are increasingly identified as causative agents of nosocomial infections but are still not under systematic genomic surveillance. In this study, using a combination of whole-genome sequencing and conjugation experiments, we sought to elucidate the genomic characteristics and transferability of resistance genes in clinical CP-ESCPM isolates from Bulgaria. Among the 36 sequenced isolates, NDM-1 (12/36), VIM-4 (11/36), VIM-86 (8/36), and OXA-48 (7/36) carbapenemases were identified; two isolates carried both NDM-1 and VIM-86. The majority of carbapenemase genes were found on self-conjugative plasmids. IncL plasmids were responsible for the spread of OXA-48 among E. hormaechei, C. freundii, and S. marcescens. IncM2 plasmids were generally associated with the spread of NDM-1 in C. freundii and S. marcescens, and also of VIM-4 in C. freundii. IncC plasmids were involved in the spread of the recently described VIM-86 in P. stuartii isolates. IncC plasmids carrying blaNDM-1 and blaVIM-86 were observed too. blaNDM-1 was also detected on IncX3 in S. marcescens and on IncT plasmid in M. morganii. The significant resistance transfer rates we observed highlight the role of the ESCPM group as a reservoir of resistance determinants and stress the need for strengthening infection control measures.

Resurgence of respiratory syncytial virus with dominance of RSV-B during the 2022-2023 season.

Background: Respiratory syncytial virus (RSV) is a common cause of upper and lower respiratory tract infections. This study aimed to explore the prevalence of respiratory syncytial virus (RSV) and other respiratory viruses in Bulgaria, characterize the genetic diversity of RSV strains, and perform amino acid sequence analyses of RSV surface and internal proteins. Methods: Clinical and epidemiological data and nasopharyngeal swabs were prospectively collected from patients with acute respiratory infections between October 2020 and May 2023. Real-time PCR for 13 respiratory viruses, whole-genome sequencing, phylogenetic, and amino acid analyses were performed. Results: This study included three epidemic seasons (2020-2021, 2021-2022, and 2022-2023) from week 40 of the previous year to week 20 of the following year. Of the 3,047 patients examined, 1,813 (59.5%) tested positive for at least one viral respiratory pathogen. RSV was the second most detected virus (10.9%) after SARS-CoV-2 (22%). Coinfections between RSV and other respiratory viruses were detected in 68 cases, including 14 with SARS-CoV-2. After two seasons of low circulation, RSV activity increased significantly during the 2022-2023 season. The detection rates of RSV were 3.2, 6.6, and 13.7% in the first, second, and third seasons, respectively. RSV was the most common virus found in children under 5 years old with bronchiolitis (40%) and pneumonia (24.5%). RSV-B drove the 2022-2023 epidemic. Phylogenetic analysis indicated that the sequenced RSV-B strains belonged to the GB5.0.5a and GB5.0.6a genotypes. Amino acid substitutions in the surface and internal proteins, including the F protein antigenic sites were identified compared to the BA prototype strain. Conclusion: This study revealed a strong resurgence of RSV in the autumn of 2022 after the lifting of anti-COVID-19 measures, the leading role of RSV as a causative agent of serious respiratory illnesses in early childhood, and relatively low genetic diversity in circulating RSV strains.

Genomic Epidemiology and Lineage Dynamics of SARS-CoV-2 in Bulgaria: Insights from a Three-Year Pandemic Analysis.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has brought about significant challenges worldwide. In this study, we present a comprehensive analysis of the genomic epidemiology and lineage dynamics of SARS-CoV-2 in Bulgaria over a three-year period. Through extensive genomic sequencing and data analysis, we investigated the evolution of the virus, the emergence of variants of concern (VOCs), and their impact on the country's pandemic trajectory. We also assessed the relationship between viral diversity and COVID-19 morbidity and mortality in Bulgaria. Our findings shed light on the temporal and spatial distribution of SARS-CoV-2 lineages and provide crucial insights into the dynamics of the pandemic in the country. The interplay between international travel and viral transmission plays a significant role in the emergence and dissemination of different SARS-CoV-2 variants. The observed proportions of exportation to various continents provide insights into the potential pathways through which these lineages spread globally. Understanding the genomic epidemiology of SARS-CoV-2 in Bulgaria is essential for formulating targeted public health strategies, enhancing vaccination efforts, and effectively managing future outbreaks.

Early Detection of the Recombinant SARS-CoV-2 XAN Variant in Bulgaria: Initial Genomic Insights into Yet Another Piece of the Growing Puzzle of Recombinant Clades.

The first recombinant SARS-CoV-2 variants were identified in 2022, causing public health concerns. The importance of recombinant variants has increased especially since the WHO designated the recombinant variant XBB and its lineages as subvariants that require monitoring on 20 November 2022. In this study, we provide the first insights into the new SARS-CoV-2 variant named XAN, a recombinant composed of Omicron sub-lineages BA.2 and BA.5. To our knowledge, this is the first report on the recombinant SARS-CoV-2 XAN variant identified in Bulgaria.

Features of Mpox infection: The analysis of the data submitted to the ID-IRI network.

Background: Mpox is a rare zoonotic disease caused by the Mpox virus. On May 21, 2022, WHO announced the emergence of confirmed Mpox cases in countries outside the endemic areas in Central and West Africa. Methods: This multicentre study was performed through the Infectious Diseases International Research Initiative network. Nineteen collaborating centres in 16 countries participated in the study. Consecutive cases with positive Mpoxv-DNA results by the polymerase chain reaction test were included in the study. Results: The mean age of 647 patients included in the study was 34.5.98.6% of cases were males, 95.3% were homosexual-bisexual, and 92.2% had a history of sexual contact. History of smallpox vaccination was present in 3.4% of cases. The median incubation period was 7.0 days. The most common symptoms and signs were rashes in 99.5%, lymphadenopathy in 65.1%, and fever in 54.9%. HIV infection was present in 93.8% of cases, and 17.8% were followed up in the hospital for further treatment. In the two weeks before the rash, prodromal symptoms occurred in 52.8% of cases. The incubation period was 3.5 days shorter in HIV-infected Mpox cases with CD4 count <200/µL, we disclosed the presence of lymphadenopathy, a characteristic finding for Mpox, accompanied the disease to a lesser extent in cases with smallpox vaccination. Conclusions: Mpox disseminates globally, not just in the endemic areas. Knowledge of clinical features, disease transmission kinetics, and rapid and effective implementation of public health measures are paramount, as reflected by our findings in this study.

Age and Gender Trends in the Prevalence of Markers for Hepatitis E Virus Exposure in the Heterogeneous Bulgarian Population.

The prevalence of hepatitis E virus (HEV) in the Bulgarian population remains underestimated. The aim of the present study was to evaluate age and gender trends in HEV prevalence in the heterogeneous Bulgarian population. Stored serum samples from blood donors and different patient sub-populations-kidney recipients (KR), patients with Guillain-Barre syndrome (GBS), Lyme disease (LD), patients with liver involvement and a clinical diagnosis other than viral hepatitis A and E (non-AE), hemodialysis (HD) and HIV-positive patients (HIV)-were retrospectively investigated for markers of past and recent/ongoing HEV infection. The estimated overall seroprevalence of past infection was 10.6%, ranging from 5.9% to 24.5% for the sub-populations evaluated, while the seroprevalence of recent/ongoing HEV infection was 7.5%, ranging from 2.1% to 20.4%. The analysis of the individual sub-populations showed a different prevalence with respect to sex. In regard to age, the cohort effect was preserved, as a multimodal pattern was observed only for the GBS sub-population. Molecular analysis revealed HEV 3f and 3e. The type of the population is one of the main factors on which the anti-HEV prevalence depends, highlighting the need for the development of guidelines related to the detection and diagnosis of HEV infection with regard to specific patient populations.

Patterns of cytokine and chemokine expression in peripheral blood of patients with COVID-19 associated with disease severity.

OBJECTIVES: Cytokine dysregulation has been proposed as one of the main culprits for severe COVID-19 and poor prognosis. We examined the parallel presence of lymphopoietic, proinflammatory, Th1, Th2, regulatory cytokines, and chemokines in the serum of 47 patients with mild, moderate, and severe COVID-19 and evaluated the association between cytokine concentrations and disease severity. METHODS: A multiplex quantitative cytokine analysis ProcartaPlex™ immunoassay was applied, using the LuminexTM 200X detection system (Invitrogen). RESULTS: The concentrations of twelve cytokines: IL-18, IFN-gamma, TNF-alpha; IL-21; IL-1alpha, IL-1beta, IL-6, IL-22; IL-10, IL-1RA; IL-7 and IFN-alpha were consistently elevated in the studied serum samples. All examined chemokines-Eotaxin, GRO-alpha, IL-8, IP-10, MCP-1, MIP-1alpha, MIP-1beta, SDF-1alpha, and RANTES, were detectable in all studied groups, confirming their importance in mediating the adaptive immune response regardless of disease severity. The serum concentrations of six mediators: IL-1beta, IL-6, IL-18, IL-10, IL-8, and IP-10, showed statistically significant differences among the groups with different disease severity. IL-6, IL-1beta, and IL-10 were more significantly elevated in severe cases while milder symptoms were associated with lower levels of IL-8 and IP-10. CONCLUSION: Overall, the studied chemokines demonstrated an associated production in acute COVID-19 infection. A strong correlation was observed between the Th1 mediators IL-18 and IL-10 and the proinflammatory IL-6 in the severe COVID-19 group. Our results indicated that severe COVID-19 was characterized by a dysregulated cytokine pattern whereby the Th1 immune response is outweighed by the immunoregulatory response, while inhibitory signals cannot balance the hyperinflammatory response.

Low prevalence of influenza viruses and predominance of A(H3N2) virus with respect to SARS-CoV-2 during the 2021-2022 season in Bulgaria.

Social distancing, mask-wearing, and travel restrictions during the COVID-19 pandemic have significantly impacted the spread of influenza viruses. The objectives of this study were to analyze the pattern of influenza virus circulation with respect to that of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Bulgaria during the 2021-2022 season and to perform a phylogenetic/molecular analysis of the hemagglutinin (HA) and neuraminidase (NA) sequences of representative influenza strains. Influenza infection was confirmed using real-time reverse transcription polymerase chain reaction in 93 (4.2%) of the 2193 patients with acute respiratory illness tested wherein all detected viruses were subtyped as A(H3N2). SARS-CoV-2 was identified in 377 (24.3%) of the 1552 patients tested. Significant differences in the incidence of influenza viruses and SARS-CoV-2 were found between individual age groups, outpatients/inpatients, and in the seasonal distribution of cases. Two cases of coinfections were identified. In hospitalized patients, the Ct values of influenza viruses at admission were lower in adults aged ≥65 years (indicating higher viral load) than in children aged 0-14 years (p < 0.05). In SARS-CoV-2-positive inpatients, this association was not statistically significant. HA genes of all A(H3N2) viruses analyzed belonged to subclade 3C.2a1b.2a. The sequenced viruses carried 11 substitutions in HA and 5 in NA, in comparison to the vaccine virus A/Cambodia/e0826360/2020, including several substitutions in the HA antigenic sites B and C. This study revealed extensive changes in the typical epidemiology of influenza infection, including a dramatic reduction in the number of cases, diminished genetic diversity of circulating viruses, changes in age, and seasonal distribution of cases.

Clinical significance and role of coinfections with respiratory pathogens among individuals with confirmed severe acute respiratory syndrome coronavirus-2 infection.

Introduction: This study aimed to determine the prevalence, viral profile, and clinical features of coinfections with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and other respiratory viruses. Methods: Nasopharyngeal samples and clinical data of 221 hospitalized patients and 21 outpatients were collected and analyzed. Real-time reverse transcription-polymerase chain reaction was used to detect SARS-CoV-2, influenza virus, respiratory syncytial virus (RSV), human metapneumovirus (HMPV), parainfluenza virus (PIV) 1,2,3, rhinovirus (RV), adenovirus (AdV), bocaviruses (BoV), and seasonal coronaviruses (OC43, 229E, NL63, and HKU1). Viral load was determined by capillary electrophoresis. Results: From November 2020 to mid-March 2022, 242 SARS-CoV-2 positive patients were tested for seasonal respiratory viruses, and 24 (9.9%) cases of coinfections were detected. The distribution of viruses involved in cases of coinfections were as follows: HMPV (n = 6; 25%), RSV (n = 4;16.7%), AdV (n = 4; 16.7%), BoV (n = 4; 16.7%), PIV3 (n = 2; 8.3%), influenza A (H3N2; n = 2; 8.3%), RV (n = 1; 4.62%), and RV+BoV (n = 1; 4.62%). The proportion of detected coinfections with SARS-CoV-2 was highest in children aged 0-5 years (59%), followed by those >65 years (33%). In specimens with detected coinfection, the viral load of influenza was higher than that of SARS-CoV-2, and the mean viral load of SARS-CoV-2 was higher than that of the other respiratory viruses. C-reactive protein (CRP) and lymphocytes count in co-infected patients >65 years of age were on average higher than in children <16 years of age (mean CRP of 161.8 ± 133.1 mg/L; 19.7 ± 3.09% vs. mean 6.9 ± 8.9 mg/L, 0.9 ± 3.1%; p < 0.01). Patients >65 years of age co-infected with SARS-CoV-2 and other respiratory viruses had longer hospital stays than those <16 years of age (mean 9 ± 3.96 days vs. 5.44 ± 1.89 days; p = 0.025). The combination of AdV and SARS-CoV-2 is fatal for patients aged >65 years. Conclusion: In patients aged >65 years, coinfection with SARS CoV-2 and other respiratory viruses, together with concomitant diseases, causes worsening of the clinical picture and complications, and can be fatal. Screening of patients with SARS CoV-2 for other respiratory viruses is needed to select appropriate treatments and prevent a fatal outcome of the disease.

Initial introduction and spread of the SARS-CoV-2 AY.4.2.1 Delta variant in Bulgaria, a genomic insight.

The evolution of the emerging SARS-CoV-2 variants carrying mutations in the spike protein raises concerns about the possibility of accelerated transmission in the ever-evolving COVID-19 pandemic worldwide. AY.4.2, a sublineage of the Delta variant, was considered a variant under investigation (VUI) and also gained the nickname "Delta Plus," due to its extra mutations, Y145H and A222V. In this study, using genomic epidemiology, we provide the first insights into the introduction of AY.4.2 in Bulgaria and the AY.4.2.1 sublineage that found larger dissemination only in Bulgaria and the United Kingdom.

Induction of humoral and cellular immune responses to COVID-19 mRNA and vector vaccines: A prospective cohort study in Bulgarian healthcare workers.

Installing efficient protective immunity by anti-SARS-CoV-2 vaccines is the only current means to overcome coronavirus disease 2019 pandemics. The cellular and humoral immune responses induced with an messenger RNA (mRNA) (BNT162b2) or with a vector (ChAdOx1nCoV-19) vaccine among Bulgarian healthcare workers (n = 123, aged 23-71 years) were studied in the course of 16 weeks after priming. Receptor-binding domain (RBD)-blocking Abs and SARS-CoV-2 RBD immunoglobulin A  (IgA) were evaluated in parallel with interferon gamma (IFNγ)-producing virus-specific T cells. Both vaccines induced RBD-blocking Abs in 100% of the participants after complete immunization while the levels of protection after a single dose largely varied (22%-98%). Advanced age had a negative impact on the level and longevity of virus-neutralizing activity induced by one dose mRNA, but not by the vector vaccine. RBD-binding IgA was detected in 100% of tested donors from the mRNA vaccine cohort, and in 67% of tested from the vector vaccine cohort, at least 1 month after completed immunization. One month after completing mRNA immunization, the number of IFNγ-producing T cells correlated significantly with the levels of RBD-specific IgA and virus-neutralizing activity induced after priming. Enumeration of circulating virus-specific IFNγ+ T cells is not recommended for evaluation of protective immunity as their detection may require longer stimulation beyond the firstmonth postimmunization. In conclusion, BNT162B2 and ChAdOx1nCoV-19 induced potent and comparable humoral and cellular anti-SARS-CoV-2 immune responses, peaking between 10 and 30 days after complete immunization. A single dose of any vaccine did not induce adequate protection in a great part of donors, making the shorter interval between mRNA vaccine doses preferable in the settings of increased risk of infection.

Short epidemiological overview of the current situation on COVID-19 pandemic in Southeast European (SEE) countries.

We are living in times where a viral disease has brought normal life in much of the world to a halt. The novel coronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) started in December 2019 in Wuhan, China initially and in a short time crossed the European borders. After mitigating the epidemic in China, Italy became one of the most COVID-19 affected countries worldwide. International travelers are important sources of infectious diseases and a possible source of epidemic. Due to its political, geographic, and cultural similarities, Italy is one of the main economic partners of Southeast European (SEE) countries. Our data show that infection in index cases in all 11 SEE countries was travel-related with Italy being a source country for 8/11 countries. After the first case identifications on February 25, the number of cases in SEE countries is continually rising reaching the total number of 15,612 with 565 fatal cases and overall case fatality ratio (CFR) of 3.6 (median 3.8, range 0.8-5.5) by April 10, 2020. At a time when the COVID-19 pandemic is approaching its peak, apart from the problems with treatment of the disease and care for critically ill patients, there are other equally important problems, such as organization of outbreak response, provision of health care, lack of hospital personnel, disruption of personal protective equipment supply chains and health care workers (HCWs) protection. But what is more important is the heroic behavior of the HCWs who are showing their humanity by disregarding their lives.