FERROPTOSIS IN CD4+ AND CD8+ T-CELLS IN THE SETTINGS OF HIV INFECTION

Introduction: Elevation of intracellular iron concentration triggers ferroptosis. Understanding the regulation and pathophysiological mechanisms of this process in HIV infection may contribute to antiretroviral therapy (cART) monitoring. Aim: To perform a correlation analysis of the intracellular labile-bound iron pool (LIP) in CD4+ and CD8+ T cells in association with CD4+, CD8+ T cells absolute count (AC) and CD4/CD8 index in HIV+ individuals on continuous cART with sustained viral suppression. Material and methods: Peripheral blood samples (Li heparin, n=34) were collected in the course of the routine immune monitoring of HIV+ individuals at four time points during 24 months. Plasma HIV viral load (VL) was determined with the Abbott Real-Time HIV-1 test (sensitivity 40 copies/ml). AC and percentage of CD4+ and CD8+ T cells were determined by direct flow cytometry (Multitest, BD Trucount, FACS Canto II). The intracellular content of LIP in CD4 and CD8 T cells (LIP CD4, LIP CD8) was measured at the beginning of the study, using acetoxymethyl ester and subsequent incubation with a chelator (Deferiprone). LIP was quantified according to the mean fluorescence intensity (MFI) (FACSCanto II, Diva 6.1.2). Results: In the settings of a higher LIP CD4 , high LIP CD8 correlated with increased CD8AC (Rho=0.70, p<0.05) up to 11 (min. 6, max. 15) months after LIP measurement., and decreased CD4/CD8 ratio correlated inversely with LIP CD8 in all consecutive measurements (Rho= -0.71, p<0.01 for all), Importantly, high LIP CD8 correlated with a lower CD4AC (Rho=-0.65, p<0.05) up to five (min.1, max.8) months after LIP measurement. Conclusion: The increased concentration of intracellular LIP in CD8 cells in HIV+cART individuals could indicate viral activity in the settings of undetectable HIV VL, directly associated with ongoing cell ferroptosis

SARS-COV-2 GENOMIC SURVEILLANCE IN BULGARIA INDICATES DIVERSE DYNAMICS DRIVEN BY MULTIPLE INTRODUCTIONS OF DIFFERENT VIRAL VARIANTS IN 2022

Background. Evolution of the emerging SARS-CoV-2 variants raises concerns about the possibility of accelerated transmission,  disease severity, diagnostic challenges, and reduced vaccine effectiveness in the ever-evolving COVID-19 pandemic worldwide. Objectives for this study were to build a comprehensive national system for monitoring and genomic surveillance of SARS-CoV-2  and to identify the introduced virus variants in the country. Methods. We analyzed SARS-CoV-2 infections in 7948 representative clinical samples collected in medical institutions in different  geographical regions of the country in 2022. Whole-genome next-generation sequencing of SARS-CoV-2 was performed on samples  from randomly selected SARS-CoV-2-positive individuals by using a modified ARTIC v3-tailed amplicon method. A bioinformatic and  phylogenetic analyses of the obtained sequences was carried out. Results. Significant dynamics was observed in the spread of viral variants in 2022, which is characterized by the introduction and  spread of multiple SARS-CoV-2 variants. The phylogenomic analysis identified a high genetic heterogeneiety composed of a total of 152 different viral clades divided into 3 main supergroups: 114 (75.0%) of which were Omicron sub-variants, 35 (23.0%) Delta sub-variants, and 3 (2.0%) recombinant forms. Conclusion. Viral variants and their sub-clades with different potentials to impact disease severity were identified and the  information was immediately published for use by decision-makers and the scientific community. The global pandemic of COVID-19  has shown the importance of molecular biological surveillance, which is an indispensable element of the modern approach in the  fight against infectious diseases

Low HIV-1 transmitted drug resistance in Bulgaria against a background of high clade diversity

Objectives To determine transmitted drug resistance (TDR) and HIV-1 genetic diversity in Bulgaria. Methods The prevalence of TDR and HIV-1 subtypes was determined in 305/1446 (21.1%) persons newly diagnosed with HIV/AIDS from 1988 to 2011. TDR mutations (TDRMs) in protease and reverse transcriptase were defined using the WHO HIV drug mutation list. Phylogenetic analysis was used to infer polymerase (pol) genotype. Results TDRMs were found in 16/305 (5.2%) persons, 11 (3.6%) with resistance to NRTIs, 5 (1.6%) with resistance to NNRTIs and 3 (0.9%) with resistance to PIs. Dual-class TDRMs were found in three (1.0%) patients and one statistically supported cluster of TDRMs comprising two individuals with subtype B infection. TDRMs were found in 10 heterosexuals, 4 MSM and two intravenous drug users. Phylogenetic analyses identified high HIV-1 diversity consisting of mostly subtype B (44.6%), subtype C (3.3%), sub-subtype A1 (2.6%), sub-subtype F1 (2.3%), sub-subtype A-like (3.6%), subtype G (0.3%), CRF14_BG (1.6%), CRF05_DF (1.3%), CRF03_AB (0.3%) and unique recombinant forms (1.3%). Conclusions We found a low prevalence of TDR against a background of high HIV-1 genetic diversity among antiretroviral-naive patients in Bulgaria. Our results provide baseline data on TDR and support continued surveillance of high-risk populations in Bulgaria to better target treatment and prevention efforts

Low HIV-1 transmitted drug resistance in Bulgaria against a background of high clade diversity

Objectives To determine transmitted drug resistance (TDR) and HIV-1 genetic diversity in Bulgaria. Methods The prevalence of TDR and HIV-1 subtypes was determined in 305/1446 (21.1%) persons newly diagnosed with HIV/AIDS from 1988 to 2011. TDR mutations (TDRMs) in protease and reverse transcriptase were defined using the WHO HIV drug mutation list. Phylogenetic analysis was used to infer polymerase (pol) genotype. Results TDRMs were found in 16/305 (5.2%) persons, 11 (3.6%) with resistance to NRTIs, 5 (1.6%) with resistance to NNRTIs and 3 (0.9%) with resistance to PIs. Dual-class TDRMs were found in three (1.0%) patients and one statistically supported cluster of TDRMs comprising two individuals with subtype B infection. TDRMs were found in 10 heterosexuals, 4 MSM and two intravenous drug users. Phylogenetic analyses identified high HIV-1 diversity consisting of mostly subtype B (44.6%), subtype C (3.3%), sub-subtype A1 (2.6%), sub-subtype F1 (2.3%), sub-subtype A-like (3.6%), subtype G (0.3%), CRF14_BG (1.6%), CRF05_DF (1.3%), CRF03_AB (0.3%) and unique recombinant forms (1.3%). Conclusions We found a low prevalence of TDR against a background of high HIV-1 genetic diversity among antiretroviral-naive patients in Bulgaria. Our results provide baseline data on TDR and support continued surveillance of high-risk populations in Bulgaria to better target treatment and prevention efforts

Prevalence and genetic characterisation of respiratory syncytial viruses circulating in Bulgaria during the 2014/15 and 2015/16 winter seasons

<p>The respiratory syncytial virus (RSV) is a leading cause of acute respiratory illnesses (ARI) in infants and young children. The objectives of this study were to investigate the RSV circulation among children aged <5 years in Bulgaria, to identify the RSV-A and RSV-B genotypes and to perform an amino acid sequence analysis of second hypervariable region (HVR2) of the G gene. During the 2014/15 and 2015/16 winter seasons, nasopharyngeal specimens of 610 children aged <5 years with ARI were tested using Real Time RT-PCR for influenza viruses, RSV, metapneumovirus, parainfluenza viruses, rhinoviruses and adenoviruses. Viral respiratory pathogens were detected in 429 (70%) out of 610 patients examined and RSV was the most frequently identified virus (26%) followed by influenza A(H1N1)pdm09 virus (14%) (<i>p</i> < .05). RSV was the most prevalent pathogen in patients with bronchiolitis (48%) and pneumonia (38%). In the 2014/15 season, RSV-A dominated slightly (53%), while in the next season RSV-B viruses prevailed more strongly (66%). The phylogenetic analysis based on the G gene indicated that all 21 studied RSV-A strains belonged to the ON1 genotype; the vast majority (96%) of the RSV-B strains were classified into BA9 genotype and only one - into BA10 genotype. All Bulgarian RSV-A and RSV-B sequences contained a 72-nt and a 60-nt duplication in the HVR2, respectively. The study showed the leading role of this pathogen as a causative agent of serious respiratory illnesses in early childhood, year-on-year fluctuations in RSV incidence, a shift from RSV-A to RSV-B subgroup dominance and relatively low genetic divergence in the circulating strains.</p

Molecular Epidemiological Analysis of the Origin and Transmission Dynamics of the HIV-1 CRF01_AE Sub-Epidemic in Bulgaria

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

Molecular Epidemiological Analysis of the Origin and Transmission Dynamics of the HIV-1 CRF01_AE Sub-Epidemic in Bulgaria

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

Transmitted HIV Drug Resistance in Bulgaria Occurs in Clusters of Individuals from Different Transmission Groups and Various Subtypes (2012–2020)

Transmitted HIV drug resistance in Bulgaria was first reported in 2015 using data from 1988–2011. We determined the prevalence of surveillance drug resistance mutations (SDRMs) and HIV-1 genetic diversity in Bulgaria during 2012–2020 using polymerase sequences from 1053 of 2010 (52.4%) antiretroviral therapy (ART)-naive individuals. Sequences were analyzed for DRM using the WHO HIV SDRM list implemented in the calculated population resistance tool at Stanford University. Genetic diversity was inferred using automated subtyping tools and phylogenetics. Cluster detection and characterization was performed using MicrobeTrace. The overall rate of SDRMs was 5.7% (60/1053), with 2.2% having resistance to nucleoside reverse transcriptase inhibitors (NRTIs), 1.8% to non-nucleoside reverse transcriptase inhibitors (NNRTIs), 2.1% to protease inhibitors (PIs), and 0.4% with dual-class SDRMs. We found high HIV-1 diversity, with the majority being subtype B (60.4%), followed by F1 (6.9%), CRF02_AG (5.2%), A1 (3.7%), CRF12_BF (0.8%), and other subtypes and recombinant forms (23%). Most (34/60, 56.7%) of the SDRMs were present in transmission clusters of different subtypes composed mostly of male-to-male sexual contact (MMSC), including a 14-member cluster of subtype B sequences from 12 MMSC and two males reporting heterosexual contact; 13 had the L90M PI mutation and one had the T215S NRTI SDRM. We found a low SDRM prevalence amid high HIV-1 diversity among ART-naive patients in Bulgaria during 2012–2020. The majority of SDRMs were found in transmission clusters containing MMSC, indicative of onward spread of SDRM in drug-naive individuals. Our study provides valuable information on the transmission dynamics of HIV drug resistance in the context of high genetic diversity in Bulgaria, for the development of enhanced prevention strategies to end the epidemic

Detailed Molecular Epidemiologic Characterization of HIV-1 Infection in Bulgaria Reveals Broad Diversity and Evolving Phylodynamics

<div><p>Limited information is available to describe the molecular epidemiology of HIV-1 in Bulgaria. To better understand the genetic diversity and the epidemiologic dynamics of HIV-1 we analyzed 125 new polymerase (<i>pol</i>) sequences from Bulgarians diagnosed through 2009 and 77 <i>pol</i> sequences available from our previous study from persons infected prior to 2007. Epidemiologic and demographic information was obtained from each participant and phylogenetic analysis was used to infer HIV-1 evolutionary histories. 120 (59.5%) persons were infected with one of five different HIV-1 subtypes (A1, B, C, F1 and H) and 63 (31.2%) persons were infected with one of six different circulating recombinant forms (CRFs; 01_AE, 02_AG, 04_cpx, 05_DF, 14_BG, and 36_cpx). We also for the first time identified infection with two different clusters of unique A-like and F-like sub-subtype variants in 12 persons (5.9%) and seven unique recombinant forms (3.5%), including a novel J/C recombinant. While subtype B was the major genotype identified and was more prevalent in MSM and increased between 2000–2005, most non-B subtypes were present in persons ≥45 years old. CRF01_AE was the most common non-B subtype and was higher in women and IDUs relative to other risk groups combined. Our results show that HIV-1 infection in Bulgaria reflects the shifting distribution of genotypes coincident with the changing epidemiology of the HIV-1 epidemic among different risk groups. Our data support increased public health interventions targeting IDUs and MSM. Furthermore, the substantial and increasing HIV-1 genetic heterogeneity, combined with fluctuating infection dynamics, highlights the importance of sustained and expanded surveillance to prevent and control HIV-1 infection in Bulgaria.</p> </div

Phylogenetic relationship of Bulgarian HIV-1 subtype B polymerase sequences.

<p>The 691-bp alignment was composed of 104 Bulgarian HIV-1 B strains and 45 subtype B reference sequences from the Los Alamos HIV database. Antiretroviral resistance-associated mutations were stripped from the alignment. The tree was rooted by using an HIV-1 subtype A1 strain as the outgroup. Tree topology was inferred using maximum likelihood analysis implemented in MEGA5. Support for each node was determined using 1,000 bootstrap replications with only values ≥70 shown. Scale bar indicates the number of nucleotide substitutions per site. Nearly identical tree topologies were also obtained with Bayesian analysis. Bulgarian sequences are shown using green branches and taxon names.</p