HIV-1 subtype CRF01_AE is the second most predominant strain in Bulgaria, yet little is known about the molecular epidemiology of its origin and transmissibility. We used a phylodynamics approach to better understand this sub-epidemic by analyzing 270 HIV-1 polymerase (pol) sequences collected from persons diagnosed with HIV/AIDS between 1995 and 2019. Using network analyses at a 1.5% genetic distance threshold (d), we found a large 154-member outbreak cluster composed mostly of persons who inject drugs (PWID) that were predominantly men. At d = 0.5%, which was used to identify more recent transmission, the large cluster dissociated into three clusters of 18, 12, and 7 members, respectively, five dyads, and 107 singletons. Phylogenetic analysis of the Bulgarian sequences with publicly available global sequences showed that CRF01_AE likely originated from multiple Asian countries, with Vietnam as the likely source of the outbreak cluster between 1988 and 1990. Our findings indicate that CRF01_AE was introduced into Bulgaria multiple times since 1988, and infections then rapidly spread among PWID locally with bridging to other risk groups and countries. CRF01_AE continues to spread in Bulgaria as evidenced by the more recent large clusters identified at d = 0.5%, highlighting the importance of public health prevention efforts in the PWID communities.

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.

The increased life expectancy of PLHIV (People Living with HIV) and the successful highly combined antiretroviral therapy (cART) poses new clinical challenges regarding aging and its co-morbid condition. It is commonly believed that HIV infection “accelerates” aging. Human immunodeficiency virus type 1 (HIV-1) infection is characterized by inflammation and immune activation that persists despite cART, and that may contribute to the development of co-morbid conditions. In this regard, we aimed to compare current cART regimens in light of premature aging to evaluate differences in their ability to reduce immune activation and inflammation in virologically suppressed patients. We studied a panel of biomarkers (IFN-γ, IL-1β, IL-12p70, IL-2, IL-4, IL-5, IL-6, IL-13, IL-18, GM-CSF, TNF-α, C-reactive protein, D-dimer, soluble CD14), which could provide a non-invasive and affordable approach to monitor HIV-related chronic inflammation. The results of the current study do not provide hard evidence favoring a particular cART regimen, although they show a less favorable regimen profile containing a protease inhibitor. Our data suggest an incomplete reduction of inflammation and immune activation in terms of the effective cART. It is likely that the interest in various biomarkers related to immune activation and inflammation as predictors of clinical outcomes among PLHIV will increase in the future.

HIV-1 subtype B is the predominant strain in Bulgaria, yet little is known about the molecular epidemiology of these infections, including its origin and transmissibility. We used a phylodynamics approach by combining and analyzing 663 HIV-1 polymerase (pol) sequences collected from persons diagnosed with HIV/AIDS between 1988–2018 and associated epidemiologic data to better understand this sub-epidemic in Bulgaria. Using network analyses at a 1.5% genetic distance threshold (d) we found several large phylogenetic clusters composed mostly of men who have sex with men (MSM) and male heterosexuals (HET). However, at d = 0.5%, used to identify more recent transmission, the largest clusters dissociated to become smaller in size. The majority of female HET and persons with other transmission risks were singletons or pairs in the network. Phylogenetic analysis of the Bulgarian pol sequences with publicly available global sequences showed that subtype B was likely introduced into Bulgaria from multiple countries, including Israel and several European countries. Our findings indicate that subtype B was introduced into Bulgaria multiple times since 1988 and then infections rapidly spread among MSM and non-disclosed MSM. These high-risk behaviors continue to spread subtype B infection in Bulgaria as evidenced by the large clusters at d = 0.5%. Relatively low levels of antiretroviral drug resistance were observed in our study. Prevention strategies should continue to include increased testing and linkage to care and treatment, as well as expanded outreach to the MSM communities.

Background/Objectives: Restored CD4 absolute counts (CD4AC) and CD4/CD8 ratio in the setting of continuous antiretroviral treatment (ART) do not exclude a low-level immune activation associated with HIV reservoirs, microbial translocation, or the side effects of ART itself, which accelerates the aging of people living with HIV (PLHIV). To delineate biomarkers of incomplete immune restoration in PLHIV on successful ART, we evaluated T-lymphocyte mitochondrial parameters in relation to phenotypic markers of immune exhaustion and senescence. Methods: PLHIV with sustained viral suppression, CD4AC >500 and CD4/CD8 ratio >0.9 on ART (n = 39) were compared to age-matched ART-naïve donors (n = 27) and HIV(–) healthy controls (HC, n = 35). CD4 and CD8 differentiation and effector subsets (CCR7/CD45RA and CD27/CD28), activation, exhaustion, and senescence markers (CD38, CD39 Treg, CD57, TIGIT, and PD-1) were determined by flow cytometry. Mitochondrial mass (MM) and membrane potential (MMP) of CD8 and CD4 T cells were evaluated with MitoTracker Green and Red flow cytometry dyes. Results: ART+PLHIV differed from HC by increased CD4 TEMRA (5.3 (2.1–8.8) vs. 3.2 (1.6–4.4), p < 0.05), persistent TIGIT+CD57–CD27+CD28– CD8+ subset (53.9 (45.5–68.9) vs. 40.1 (26.7–58.5), p < 0.05), and expanding preapoptotic TIGIT–CD57+CD8+ effectors (9.2 (4.3–21.8) vs. 3.0 (1.5–7.3), p < 0.01) in correlation with increased CD8+ MMP (2527 (1675–4080) vs.1477 (1280–1691), p < 0.01). These aberrations were independent of age, time to ART, or ART duration, and were combined with increasing CD4 T cell MMP and MM. Conclusions: In spite of recovered CD4AC and CD4/CD8 ratio, the increased CD8+ MMP, combined with elevated markers of exhaustion and senescence in ART+PLHIV, signals a malfunction of the CD8 effector pool that may compromise viral reservoir latency.

By 2022, Bulgaria’s National Reference Laboratory had confirmed 4024 HIV cases. We analyzed 132 pol gene sequences to characterize the molecular epidemiology of HIV-1 subtypes A1, A6, and A7 (2001–2022). A1 accounted for 50.0% (66/132) of cases, increasing after 2014, with peaks in 2019 and 2022. A6 comprised 48.5% (64/132), dominating from 2005 to 2014 before stabilizing. A7 was rare (1.5%, 2/132), detected in 2003 and 2011. Transmission patterns varied: A1 was linked to men who have sex with men (MSM) (62.1%), while A6 was primarily heterosexual (HET) (82.8%) with a balanced gender distribution (56.3% male, 43.8% female). Resistance mutations were identified in 29.6% of cases, with A6 showing higher rates of nucleoside reverse transcriptase inhibitor (NRTI) (20.3%) and non-nucleoside reverse transcriptase inhibitor (NNRTI) (7.8%) resistance than A1. Phylogenetic analysis revealed that 13 Bulgarian sequences (9.8%) were involved in transmission clusters, including 10 (7.6%) from sub-subtype A1 and 3 (2.3%) from sub-subtype A6, highlighting distinct genetic diversity and transmission patterns. Despite significant migration from Ukraine in 2022, A6 prevalence remained unchanged, suggesting localized transmission dynamics. These findings highlight a shifting HIV-1 sub-subtype distribution in Bulgaria and emphasize the need for targeted prevention, diagnosis, and treatment strategies tailored to the evolving molecular landscape.