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.

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 C_t 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.

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.

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.

The COVID-19 pandemic is associated with high morbidity and significant mortality worldwide. The objective of this study was to track the circulation pattern of SARS-CoV-2 in Bulgaria over three consecutive years (2020-2022) and to analyze the involvement of SARS-CoV-2 in cases of co-infections. A total of 98 247 clinical samples were tested for SARS-CoV-2 using a Real-Time RT-PCR method and 25.2% of them were positive. The positive rate for SARS-CoV-2 was greater among hospitalized patients compared to outpatients (p<0.05). Approximately 48.3% of all SARS-CoV-2-positive cases were male and 51.7% were female (p<0.05). SARS-CoV-2 positivity was highest in the group of oldest adults (≥65 years) (average 40.6%), and lowest in the group of youngest children (0-5 years) (average 9.4%). Several peaks in the spread of SARS-CoV-2 infections were observed. Among the 1 463 SARS-CoV-2 positive clinical samples examined for the presence of other respiratory viruses, 109 (7.5%) cases of co-infections were found. The greatest variety of co-infections with SARS-CoV-2 and other respiratory viruses was detected during the Omicron wave. Surveillance of SARS-CoV-2 is important to continue in the future in order not to miss the emergence of new genetic variants with increased infectivity, virulence or immune escape.

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 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. Regular multiplex testing is important for prompt treatment and targeted antibiotic use.