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.

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.

The coast of the Bulgarian Black Sea is a popular summer holiday destination. The Dam of Iskar is the largest artificial dam in Bulgaria, with a capacity of 675 million m³. It is the main source of tap water for the capital Sofia and for irrigating the surrounding valley. There is a close relationship between the quality of aquatic ecosystems and human health as many infections are waterborne. Rapid molecular methods for the analysis of highly pathogenic bacteria have been developed for monitoring quality. Mycobacterial species can be isolated from waste, surface, recreational, ground and tap waters and human pathogenicity of nontuberculose mycobacteria (NTM) is well recognized. The objective of our study was to perform molecular analysis for key-pathogens, with a focus on mycobacteria, in water samples collected from the Black Sea and the Dam of Iskar. In a two year period, 38 water samples were collected—24 from the Dam of Iskar and 14 from the Black Sea coastal zone. Fifty liter water samples were concentrated by ultrafiltration. Molecular analysis for 15 pathogens, including all species of genus Mycobacterium was performed. Our results showed presence of Vibrio spp. in the Black Sea. Rotavirus A was also identified in four samples from the Dam of Iskar. Toxigenic Escherichia coli was present in both locations, based on markers for stx1 and stx2 genes. No detectable amounts of Cryptosporidium were detected in either location using immunomagnetic separation and fluorescence microscopy. Furthermore, mass spectrometry analyses did not detect key cyanobacterial toxins. On the basis of the results obtained we can conclude that for the period 2012–2014 no Mycobacterium species were present in the water samples. During the study period no cases of waterborne infections were reported.

Background: Pneumocystis pneumonia (PCP) commonly affects immunocompromised individuals, whereas in immunocompetent persons, it occurs relatively rarely, and in most cases, the Pneumocystis infection is detected as an asymptomatic colonization. The present study aimed to establish the prevalence of Pneumocystis jirovecii infection in human hosts with different immune status (immunocompromised and immunocompetent), using molecular diagnostic methods, and to compare their diagnostic value with that of classical staining methods. Methods: We used the collected-to-this-moment data from a prospective study on the prevalence of pneumocystosis among the Bulgarian population. Clinical specimens (including throat secretions, induced sputum, tracheal aspirates, and bronchoalveolar lavage) collected from 220 patients suspected of PCP (153 immunocompetent and 67 immunocompromised patients) were examined with microscopic staining methods and real-time PCR for detection of P. jirovecii. Results: DNA of the pathogen was detected in 38 (17%) specimens (32 immunocompromised patients and 6 immunocompetent subjects). From all 220 clinical samples examined by staining methods, only five (2%) P. jirovecii cysts were detected by the Gomori stain. All patients with PCP were treated with trimethoprim-sulfamethoxazole, but in ten of them (HIV-positive patients), the disease had a fatal outcome. Conclusions: This study is the first in Bulgaria including the main available laboratory methods for diagnosis of human pneumocystosis. Regarding the etiological diagnosis of PCP, in our study the sensitivity of real-time PCR was higher compared to the staining methods. The choice of a method for sample collection and examination has an important role in the efficiency of the laboratory diagnostics.

Background/Objectives: The increasing prevalence of Mycoplasma genitalium infections with macrolide-resistance, causing high azithromycin failure rates, is a major concern internationally. In response to this challenge, diagnostics that simultaneously detect M. genitalium and genetic markers for macrolide-resistance enable the therapy to be individually tailored, i.e., to implement resistance-guided therapy (RGT). This study aimed to evaluate patient treatment outcomes of M. genitalium therapy, guided by a macrolide-resistance assay in Bulgaria. Methods: Consecutively referred M. genitalium infection cases (n = 17) were analyzed for macrolide-resistance mutations (MRMs) and specific antimicrobial treatment was recommended accordingly (MRMs-negative infections received azithromycin and MRMs-positive infections received moxifloxacin). The treatment outcome based on test-of-cure was recorded, and the treatment failure rates and time to achieve a microbiological cure were compared to treatment outcomes in patients treated before the implementation of RGT. Results: Among patients given RGT (n = 17), the overall treatment failure rate was 1/17 (5.9%). This was significantly lower than the rate (47.6%) observed in patients treated pre-RGT (p = 0.002). The time to achieve a microbiological cure was 29.4 days (CI 24.5–34.3), compared to 45.2 days (CI 36.5–53.7) pre-RGT (p = 0.001). Conclusions: The implementation of M. genitalium diagnostics with macrolide-resistance detection improved treatment outcomes in Bulgaria, with significantly lower treatment failure rates and reduced time to achieve a microbiological cure. In light of the limited treatment options and concerns about their decreasing efficacy in response to misuse and overuse, a diagnostic macrolide-resistance assay is critical to direct the appropriate first-line treatment, to maintain the efficacy of antimicrobial treatment (antibiotic stewardship) and to minimize the spread of antimicrobial resistance.

Introduction: An effective vaccination strategy requires monitoring serotype changes by geography and age. This study analyzed Streptococcus pneumoniae serotypes in healthy children under 6 years of age vaccinated with PCV10 in Bulgaria from October 2021 to May 2025. Methods: A total of 569 children were screened for the lytA and cpsA genes viareal-time polymerase chain reaction (real-time PCR). Positive samples were typed using relevant kits, and 76 serotypes/serogroups of S. pneumoniae were identified. Results: Nasopharyngeal swabs from 232 children (40.8%) were found to carry S. pneumoniae, and a total of 255 serotypes were detected, with 19B/19C (17.2%), 6C (10.7%), and 15B/15C (9.8%) being the most prevalent. Of these, 91 serotypes (15.9%) were included in at least one vaccine, while the remaining 164 serotypes (25.4%) were not. The carriage rate reduced to 22% in 2023 but increased to 47% in 2024. Overall, younger children had lower carriage rates (p < 0.05), with serotype 6C being more common in children under 12 months of age (25%). Approximately 9.1% of pneumococcal carriage cases involved co-detected serotypes, with significantly higher co-detection rates for 19B/19C, 15B/15C, 10B, 10F/C, 23B, 7C/40, 23A, and 24A compared with mono-detection rates (p < 0.05). Conclusions: 19B/19C, 6C, 15B/15C, and 19A were identified as the main serotypes. Children over 3 years of age were also more likely to carry multiple pneumococci. These findings emphasize the need to reassess childhood vaccination strategies to curb the spread of antibiotic-resistant serotypes.